|Cancer||Cell death||Cell cycle||Cytoskeleton||Exo/endocytosis||Differentiation||Division||Organelles||Signalling||Stem cells||Trafficking|
Switching from bone marrow-derived neurons to epithelial cells through dedifferentiation and translineage redifferentiation
Yang Liu*†, Xiaohua Jiang†1, Mei Kuen Yu†, Jianda Dong†‡, Xiaohu Zhang†, Lai Ling Tsang†, Yiu Wa Chung†, Tingyu Li*1 and Hsiao Chang Chan†
*Childrens Hospital, Chongqing Medical University, Chongqing, Peoples Republic of China, †Epithelial Cell Biology Research Center, Key Laboratory for Regenerative Medicine of Ministry of Education of China, School of Biomedical Sciences, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong, and ‡Key Laboratory of Reproduction and Genetic of Ningxia Hui Autonomous Region, Key Laboratory of Fertility Preservation and Maintenance of Ningxia Medical University and Ministry of Education of China, Yinchuan, Peoples Republic of China
While the ability of stem cells to switch lineages has been suggested, the route(s) through which this may happen is unclear. To date, the best characterized adult stem cell population considered to possess transdifferentiation capacity is BM-MSCs (bone marrow mesenchymal stem cells). We investigated whether BM-MSCs that had terminally differentiated into the neural or epithelial lineage could be induced to transdifferentiate into the other phenotype in vitro. Our results reveal that neuronal phenotypic cells derived from adult rat bone marrow cells can be switched to epithelial phenotypic cells, or vice versa, by culture manipulation allowing the differentiated cells to go through, first, dedifferentiation and then redifferentiation to another phenotype. Direct transdifferentiation from differentiated neuronal or epithelial phenotype to the other differentiated phenotype cannot be observed even when appropriate culture conditions are provided. Thus, dedifferentiation appears to be a prerequisite for changing fate and differentiating into a different lineage from a differentiated cell population.
Key words: bone marrow mesenchymal stem cell, dedifferentiation, epithelial differentiation, neuronal differentiation, redifferentiation
Abbreviations: BM-MSCs, bone marrow mesenchymal stem cells, EGF, epidermal growth factor, ENaC α, epithelial Na+ channel α subunit, ENaC γ, epithelial Na+ channel γ subunit, MAP-2, microtube-associated protein-2, NF-M, neurofilament-M, NIM, neuronal induction media, rMSCs, rat mesenchymal stem cells
1Correspondence may be addressed to either author (email email@example.com or firstname.lastname@example.org).
Part of a series marking the 70th birthday of the Cell Biology International Editor-in-Chief Denys Wheatley
During the succession of early embryogenesis, distinct lineages emerge from pluripotent cells and progress to more restricted cells, giving rise to the specialized cells of different organs and tissues. Cells have been thought to only progress in one direction along these distinct lineage pathways and to be unable to switch tracts. Recent studies in stem cell research, however, have indicated that certain mammalian stem cells, even from adults, may be more plastic than we previously thought in that they maintain the ability for multilineage cell differentiation and may turn into cells of unrelated lineages in response to environmental cues (Ferrari et al., 1998; Bjornson et al., 1999; Brazelton et al., 2000; Clarke et al., 2000; Lagasse et al., 2000; Mezey et al., 2000; Orlic et al., 2001; Frisen, 2002). Stem cells capable of such plasticity have been isolated from brain (Clarke et al., 2000), bone marrow (Jiang et al., 2002), skin (Joannides et al., 2004), fat (Zuk et al., 2002), skeletal muscle (Alessandri et al., 2004), umbilical cord blood (Kadivar et al., 2006) and other visceral organs. However, the underlying mechanisms through which lineage conversion may occur are poorly understood. Do stem cells switch their phenotype directly from one cell type to another? Do differentiated cells dedifferentiate first to a primitive cell type before committing to a different lineage? Answers to these questions will definitely enhance our understanding of how stem cells maintain their multipotentiality and meanwhile control their commitment and differentiation (Song et al., 2006).
A given cell can change its developmental fate and differentiate into a cell type of a distinct lineage in one of three ways (Cobaleda and Busslinger, 2008). First, an uncommitted progenitor may change its normal developmental potential, which results in lineage diversion and subsequent differentiation into a new cell type. Secondly, a committed cell of one lineage may lose its lineage identity and undergo direct transdifferentiation to a committed cell type of another lineage. Thirdly, a committed cell of one lineage may dedifferentiate back to uncommitted progenitors followed by subsequent differentiation along another lineage. To test these possibilities, we established cultures of bone marrow-derived (rats) pluripotent stem cells, which could be induced to differentiate into neuronal or epithelial phenotypic cells by defined culture conditions. The switching of committed phenotype from one to the other was attempted at different stages of differentiation, as depicted in Figure 1, and succeeded only when differentiated neuronal or epithelial phenotypic cells were allowed to go through dedifferentiation followed by subsequent redifferentiation into the other phenotype with appropriate culture conditions.
2. Materials and methods
2.1. Establishment of primary monoclonal rMSCs (rat mesenchymal stem cells)
rMSCs were isolated from the femurs and tibias of 4-week-old SD (Sprague–Dawley) rats as previously described (Woodbury et al., 2000; Li et al., 2004). To obtain monoclonal MSCs, cells were diluted to 10 cells/ml and then seeded in 96-well plate. The wells with a singe cell were labelled, and single cells were expanded in DMEM (Dulbecco's modified Eagle's medium)/F12 supplemented with 10% FBS (fetal bovine serum) (full media) for about 4 weeks to achieve MSC clones.
2.2. Neural differentiation of rMSCs
Monoclonal rMSCs of passages 12–25 were grown on either chamber slide (Nunc Lab-Tek II-CC2 chamber slide system, four wells, 1×104/well) or 75 cm2 plastic flasks (1×106) in full media for 24 h. The media were then replaced with preinduction media consisting of DMEM/F12/10% FBS/10−7 M ATRA (all-trans-retinoic acid) and 10 ng/ml bFGF (basic fibroblast growth factor) (Li et al., 2004). To initiate neural differentiation, the preinduction media were removed, and the cells were washed with HBSS and transferred to modified NIM (neuronal induction media) composed of DMEM/F12/2% DMSO/200 μM BHA (butylated hydroxyanisole)/25 mM KCl/2 mM valproic acid/10 μM forskolin/1 μM hydrocortisone/5μg/ml insulin.
2.3. Epithelial differentiation of rMSCs
Monoclonal rMSCs (1×106) were plated onto the apical compartment of a permeable support with a transparent transwell membrane (Corning, transwell membrane insert, growth surface area 4.67 cm2, pore size 0.4 μm). Cells were grown in full media at 37°C in 95% air/5% CO2 for 4 days. For some experiments, cells were grown in full media for 24 h followed by epithelial induction media [EM; DMEM/F12/0.2% FBS/0.25 U/ml insulin/10 μg/ml transferrin/20 ng/ml EGF (epidermal growth factor), 10−7 M ATRA] for another 72 h (Plateroti et al., 1997).
2.4. Induction of translineage differentiation
To induce translineage differentiation from neuron to epithelial cells, MSC-derived neurons were treated as follows: Method 1: after neural induction for 24 h, NIM was replaced with SF (serum-free) DMEM/F12 or EM, and the cells were further cultured for 4 days. Method 2: NIM was replaced with full media, and the cells were cultured for another 4 days. Method 3: 24 h after neural induction, cells were trypsinized and transferred to a permeable support (1×106/4.67 cm2) in EM for up to 4 days.
To induce translineage differentiation from epithelial cells to neurons, epithelial cells grown on a permeable support for 4 days were scraped off and re-plated on a chamber slide (1×104 cells/well), in NIM for neural induction.
2.5. Induction of dedifferentiation and redifferentiation
After being cultured in NIM for 24 h, rMSC-derived neurons were washed with HBSS and re-incubated in full media for another 24 h. Re-incubation in full media rapidly reverted rMSC-derived neurons to characteristic mesenchymal morphology. Cells were then trypsinized and transferred onto a permeable support (1×106/4.67 cm2) in EM for 4 days to achieve epithelial phenotype. Then, epithelial layer was scraped off and plated onto a chamber slide in full media for another 4 days to allow for dedifferentiation and proliferation before neural induction.
Cultured cells on chamber slides were fixed with 4% paraformaldehyde in PBS. Cells grown on the apical compartment of permeable support for 4 days were washed in PBS three times and embedded into OCT (Tissue-Tek OCT, VWR International). Samples were cut into 7-μm sections with a cryostat (Leica CM1900). Cells or cryosections were blocked in 5% normal serum at room temperature for 1 h and followed with primary antibodies (MAP-2, 1:200, Chemicon; NF-M, 1:100, Cell Signaling Technology; Cytokeratin 19, 1:200, Santa Cruz; ENaC γ (epithelial Na+ channel γ subunit), 1:200, Millipore) at 4°C overnight. Then, cells or sections were washed with PBS and incubated with secondary antibodies (Alexa Fluor® 568 goat anti-rabbit IgG, Alexa Fluor® 488 goat anti-mouse IgG, Alexa Fluor® 488 rabbit anti-goat IgG, Invitrogen). Samples were counterstained with DAPI (4′,6-diamidino-2-phenylindole; Invitrogen), examined with an Olympus Eclipse 80i Upright Microscope and photographed using a SPOT RT-SE6 1.4MP Slider camera on the system (Diagnostic Instruments, Inc.).
The cells were scraped from the flasks or the apical compartment of permeable supports, and total RNA was extracted from the cells using TRIzol (Invitrogen). RNA (5 μg) was reverse transcribed using M-MLV Reverse Transcriptase (USB, GE Healthcare) in a 20-µl reaction, and 2 μl of reverse-transcription product was amplified by PCR using standard protocols. Primer sequences, annealing temperatures and cycle numbers are provided as follows: ZO-1 (sense: 5′-CGGAACTATGACCATCGCCTAC-3′, antisense: 5′-GCCTGTACCTGTTGTGCACC-3′, 58°C, 28 cycles); ENaC α (epithelial Na+ channel α subunit) (sense: 5′-TCACTTCAGCACATCTTCCAC-3′, antisense: 5′-TTGGTCGTTTCCCGGGAACGT-3′, 60°C, 28 cycles); ENaC γ (sense: 5′-AAATCCACAGAAGGACCTGAT-3′, antisense: 5′-TACAGATACTCTCAGTTCAAAGAC-3′; 60°C, 28 cycles). The intensities of ZO-1 and ENaC subunits were normalized to that of GAPDH (340 bp), which was amplified simultaneously.
3.1. Induction of neural differentiation
rMSCs derived from a single cell displayed uniform fibroblast-like morphology (Figure 2a). To promote the differentiation of rMSCs towards a neural lineage, we cultured subconfluent rMSCs in ATRA- and bFGF-supplemented serum-containing medium for 24 h as previously described (Li et al., 2004). The medium was then replaced with modified NIM as described in the Materials and methods section. Upon neural induction, some rMSCs rapidly adopted a neural-like morphology within 3 h. Such a response was significantly enhanced in the next 24 h, at which about 90% of cells displayed a neural-like phenotype with contracted bipolar and multipolar cell bodies and process-like extensions (Figure 2b). To further characterize the neural-like cells, we stained the cultures with two neuronal markers, MAP-2 (microtube-associated protein-2) and NF-M (neurofilament-M), which specifically target microtube-associated and intermediate filament proteins of neurons (Brazelton et al., 2000). In line with the morphological changes, the expression of MAP-2 and NF-M was undetectable in uncommitted rMSCs, whereas it significantly increased after neural induction for 24 h. As shown in Figures 2(c) and 2(d), the majority of the induced cells (>90%) were either MAP-2- or NF-M-positive. Three individual clones of rMSCs were examined for neural differentiation with similar results observed (data not shown). Consistent with previous reports (Woodbury et al., 2000; Woodbury et al., 2002), we have shown that rMSCs can be induced to differentiate into neural lineage in vitro by using the same protocol. Of note, the differentiation efficiency was significantly enhanced (>90%) by adding ATRA, a critical differentiation factor, to the preincubation media (Li et al., 2004).
3.2. Epithelial differentiation of rMSCs
For epithelial induction, 1×106 rMSCs were plated onto the apical compartment of a permeable support (Figure 3b), which has been used for growing a variety of polarized epithelia in culture (Chan et al., 1997; Bickenbach and Stern, 2005). The cells were grown in full medium for 4 days or full medium followed with EM as described in the Materials and methods section. As shown in Figures 3(a)–3(c), rMSCs presented a monolayer culture of epithelia-like cuboidal cell shape after 4 days on the microporous membrane. In addition, immunofluoresence staining detected the expression of various epithelial markers, including ENaC γ (Figure 3d), E-cadherin (Figure 3e) and cytokeratin 19 (Figure 3f) in the differentiating rMSCs. Interestingly, epithelial markers were virtually undetectable in rMSCs continually maintained on chamber slides (data not shown). Consistent with these observations, RT-PCR results showed that the expression of tight junction protein ZO-1 and different subunits of ENaC was significantly induced in the cells grown on permeable supports with full medium or EM, compared with their counterparts grown on flask, respectively (Figures 3g–3g′).
Taken together, these results suggest that rMSCs can be induced to differentiate into epithelial phenotype when grown on permeable supports, an environment suitable for forming polarized epithelia. We also find that cells grown on the permeable support with EM exhibit higher expression of epithelial markers, indicating that certain growth factors, i.e. EGF, insulin promote epithelial induction. In addition, a plating number of 1×106 cells per permeable support (4.67 cm2) appear to be critical for the establishment of epithelial monolayer.
3.3. Infeasibility of translineage differentiation in vitro
Thus far, we demonstrated that rMSCs could be induced to differentiate towards either neural or epithelial lineages by defined culture conditions. We then aimed to test whether the committed stem cells possess an intrinsic capacity to generate derivatives outside its already established differentiation path by transdifferentiation (Figure 1b). To identify conditions that may promote transdifferentiation of MSC-derived neural cells towards an epithelial lineage, we cultured rMSC-derived neurons either under SF conditions in DMEM/F-12 or EM or in the presence of 10% FBS in DMEM F-12 for 4 days. Our results showed that MSCs exhibiting neural phenotype failed to show any traits of epithelial commitment as detected by both morphological and immunohistochemical methods in response to SF DMEM/F-12 or EM with or without permeable support (Figures 4a–4g).
Specifically, if neuronal phenotypic cells were directly transferred to a microporous membrane in EM, they formed clumps with disappearance of recognizable neuronal characteristics and remained unattached to the permeable support for up to 4 days without forming an epithelial layer (Figure 4c). However, we did observe that neural cells rapidly resumed stem cell morphological characteristics after 24 h in serum-containing medium (Figure 4b), suggesting dedifferentiation of neuronal phenotypic cells in the presence of serum (Larsson et al., 1985; Woodbury et al., 2002).
Next, we tested the possibility of switching rMSC-derived epithelial cells into neural lineages in vitro. Cells grown on permeable supports exhibiting epithelial phenotype were transferred to culture apparatus in NIM for neural induction. As photographed in Figure 4(h), the cells formed clusters without any development of morphological characteristics of neural phenotype and died 16 h later. Taken together, these results suggest that translineage differentiation, switching from one differentiated phenotype directly to another distinct phenotype, is unlikely, at least for neuronal and epithelial phenotypic cells under the presently defined conditions.
3.4. Switching between neural and epithelial cells: dedifferentiation and redifferentiation
Recent studies have shown that differentiated lymphocytes can be redirected to other lineages through dedifferentiate back to uncommitted progenitors followed by subsequent differentiation along another lineages (Cobaleda et al., 2007; Cobaleda and Busslinger, 2008). We tested whether switching from neurons to epithelial cells, or vice versa, could be possible through dedifferentiation and redifferentiation. Withdrawal of NIM from MSC-derived neurons (Figure 5a) elicited process retraction and reversion of morphology to flat cells within 24 h (Figure 5c), which shared characteristics with uncommitted MSCs. Immunohistochemical staining verified that these cells expressed MSC marker vimentin (Figure 5d), but not neuronal marker MAP-2 (Figure 5e) and NF-M (Figure 5f), indicating the mesenchymal phenotype of the cells. The dedifferentiated cells were allowed to proliferate for 24 h and then transferred onto permeable supports in EM for epithelial induction. After 4 days on permeable supports, the dedifferentiated MSCs formed a tight monolayer (Figure 5g) and assumed epithelial characteristics with expression of ENaC subunits and ZO-1 (Figures 5o–5o′).
Subsequently, the epithelial cells were scrapped off the support membrane and transferred to serum-containing DMEM/F-12. After 4 days of dedifferentiation, the cells were re-induced to neural lineage under growth conditions identical to those used before. As shown in Figures 5(h)–5(n), the reverted rMSCs underwent neural phenotypic changes with corresponding MAP-2 and NF-M immunoreactivity after the process of dedifferentiation and redifferentiation. Thus, we have demonstrated that from the same population of stem cells, neuronal or epithelial phenotypic cells could be derived and that the switching between the two was possible only through dedifferentiation and redifferentiation.
Transdifferentiation refers to the process in which stem cells of a certain lineage differentiate into cell types of a different lineages across embryonic germ layers or the process in which fully differentiated cells switch their phenotype and acquire characteristics of other cell types within or beyond their original lineages. The present study was designed to explore the possible routes through which stem cell transdifferentiation occurs by an in vitro differentiation and dedifferentiation culture system using monoclonal rMSCs. Our results revealed the following: (i) rMSCs can be transdifferentiated into either neural or epithelial lineage in vitro; (ii) direct translineage differentiation from neural to epithelial or vice versa is not feasible in vitro; and (iii) the switching of neural to epithelial phenotype from one to the other can only be achieved through dedifferentiation followed by subsequent redifferentiation into the other phenotype with appropriate culture conditions.
The observation that differentiation and dedifferentiation can be triggered upon the addition or withdrawal of inducing factors suggests that the stemness and differentiation states of stem cells must be exquisitely regulated. In our study, it is clear that environment, rather than intracellular milieu, plays a crucial role in determining the fate of the cell. For example, cells grown on the permeable support, with apical and basolateral compartmental environment similar to physiological conditions for epithelial cells tend to assume epithelial phenotype, while the same cell population could be switched to neuronal phenotype by exposure to different substrates (e.g. glass/plastic) in NIM. The present study has also excluded the confounding factor of using heterogeneous population of MSCs by monoclonal strategy. We observed transdifferentiation of MSCs, which were derived from a single cell, into either neural cells or epithelial cells.
In addition, we have demonstrated that adult MSCs could be induced to dedifferentiate into a primitive stem cell-like population, upon withdrawal of extrinsic stimuli after their lineage commitment. Moreover, the stem cell plasticity lies in their ability to dedifferentiate to more primitive stem cells, which could be reprogrammed to redifferentiate along another lineage in response to environmental cues. Although it is possible that the transdifferentiation between the neural and epithelial cells was a result of stochastic expansion of certain progenitor or committed cells during the dedifferentiation and redifferentiation process, it is unlikely to be the case here. First, we and others have demonstrated that the majority of the homogeneous rMSCs-derived neural cells can dedifferentiate in the absence of continuous induction (Figure 4 and Woodbury et al., 2002). Secondly, there was a dramatic phenotypic loss of differentiated cells as detected by immunostaining (Figure 4). Thirdly, the inability of neuronal phenotypic cells to transdifferentiate into epithelial phenotypic cells directly, or vice versa, also excludes heterogeneous possibility. If the lineage conversion was due to mixed cell populations, switching phenotypes should have also been observed under direct translineage differentiation. Taken together, these results imply that dedifferentiation is very likely a necessary step for differentiated stem cells to pursue transdifferentiation, similar to the events occurring during the regeneration process in newts after limb amputation (Tsonis, 2000). The importance of this dedifferentiation and redifferentiation phenomenon is reinforced by the recent study showing that human β-cells in culture can dedifferentiate into a vimentin-expressing mesenchymal-like cell type and that these cells have significant potential for proliferation and redifferentiation (Russ et al., 2008), which raises the intriguing possibility that the mechanism of dedifferentiation and redifferentiation contributes to regeneration and repair processes in mammals as well.
Therefore, the present study has provided clear evidence for stem cell plasticity, where the possibility of heterogeneous contamination has been ruled out. It remains to be elucidated how cell fate can be reprogrammed and why only stem cells can be reprogrammed to switch lineage. The presently defined culture conditions for lineage conversion provide a useful experimental model for the studies of molecular mechanisms underlying stem cell plasticity.
Yang Liu was in charge of the collection and/or assembly of data, data analysis and interpretation, manuscript writing and final approval of the manuscript. Xiaohua Jiang was responsible for the conception and design, data analysis and interpretation, manuscript writing and final approval of the manuscript. Mei Kuen Yu, Jianda Dong, Xiaohu Zhang, Lai Ling Tsang, Yiu Wa Chung were in charge of the collection and/or assembly of data and the final approval of the manuscript. Tingyu Li was responsible for the conception and design, data analysis and interpretation and final approval of the manuscript. Hsiao Chang Chan was in charge of the conception and design, data analysis and interpretation, manuscript writing, financial support and final approval of the manuscript.
The work was supported, in part, by the
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Received 12 July 2010; accepted 28 July 2010
Published online 24 September 2010, doi:10.1042/CBI20100516
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ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
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