Trpm7 and magt1 in the osteogenic differentiation of human mesenchymal stem cells in vitro

ABSTRACT Mesenchymal stem cells are fundamental for bone formation and repair since they respond to microenvironmental stimuli by undergoing osteogenic differentiation. We show that the

kinase and cation channel TRPM7 and the magnesium transporter MagT1 have a role in harmonizing the osteogenic differentiation of human mesenchymal stem cells. TRPM7 and MagT1 are upregulated

in osteogenic differentiation and silencing either one accelerates osteogenic differentiation, partly through the activation of autophagy. Intriguingly, similar results were obtained when

the cells were cultured under magnesium deficient conditions. These results underpin the contribution of magnesium, TRPM7 and MagT1 to autophagy and osteoblastogenesis. SIMILAR CONTENT BEING

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MTORC2/AKT-473/RUNX2 AXIS Article Open access 07 June 2022 INTRODUCTION The bone is a metabolically active tissue that is continuously remodeled in development and throughout life to repair

microdamages and adjust its architecture to changing mechanical needs1. This dynamic process relies on the coordinated and timely balance between bone resorption by osteoclasts and bone

formation by osteoblasts. In particular, osteoblasts arise from bone marrow mesenchymal stem cells (MSC), rare pluripotent cells that activate the genetic program leading to

osteoblastogenesis in response to specific stimuli from the microenvironment2. There is a growing interest in MSC because of their use in cell-based therapy as a treatment strategy in

orthopedics. It is therefore essential to disclose the molecular events involved in their differentiation into osteoblasts. Both chemical and physical cues modulate the fate commitment of

bone MSC3. In particular, upon exposure to shear forces MSC exhibit dose- and time-dependent changes in gene expression that lead to the acquisition of an osteogenic phenotype4. Recently,

Transient Receptor Potential Melastatin 7 (TRPM7), a dual-function kinase and cation channel, has been shown to mediate the osteogenic differentiation of murine MSC in response to shear

stress5. Accordingly, in these cells TRPM7 directly senses membrane tension and is involved in mechanotransduction6. Moreover, TRPM7 is fundamental for murine MSC survival7. While TRPM7 is

implicated in the transport of divalent cations, primarily calcium (Ca) and magnesium (Mg)8 both crucial components of the bone, Mg transporter 1 (MagT1), which is expressed in all human

tissues, selectively transports Mg across the plasma membrane9. Rather little is known about the expression and the role of MagT1 in bone. Rat MSC cultured on Zn/Mg surfaces, which promote

osteogenesis, significantly upregulate MagT1 gene expression10. In rat MSC, silencing _MagT1_ blunts osteogenic differentiation11. Since both MagT1 and TRPM7 contribute to the maintenance of

Mg homeostasis at the cellular level, it should be recalled that Mg, the fourth most abundant metal ion in the body mostly stored in the skeleton12, plays a crucial role in bone metabolism

and in the regulation of bone cell functions13. A recent report shows that Mg deprivation as well as mesendogen, an inhibitor of TRPM7, robustly enhance mesoderm and definitive endoderm

differentiation of embryonic stem cells14. On these bases, we investigated the expression and the role of _TRPM7_ and _MagT1_ in human MSC (hMSC) induced to differentiate into osteoblasts by

exposure to an osteogenic cocktail. We evaluated the expression of some osteogenic differentiation markers. In particular, we focused on Runt-related transcription factor 2 (_RUNX2_) and

collagen type I (_COL1A1_). _RUNX2_, the master regulator of osteogenesis15, acts early to commit MSC to the osteochondral lineages and then induces the expression of _COL1A1_, which is

crucial for the osteogenic phenotype. The rational for studying the expression of _RUNX2_ and _COL1A1_ resides in our recent findings showing that if the upregulation of _RUNX2_ is not

accompanied by the increase of _COL1A1_, calcium deposition does not occur and this prevents hMSC full differentiation into osteoblasts16. A connection exists between osteogenesis and

autophagy, an evolutionary conserved self-degradative system that delivers cytoplasmic constituents to the lysosomes17. _In vivo_, the deletion of FIP200, an essential component of the

autophagosome complex, suppresses autophagy and causes osteopenia in mice by inhibiting osteoblast differentiation18. Similarly, knocking down autophagy-related gene _ATG5_, a component of

the autophagosome, reduces bone mineralization19. _In vitro_, the autophagy proteins ATG7 and beclin 1 are required for mineralization in an osteoblastic cell line19. Therefore, we evaluated

the activation and the role of autophagy in hMSC after inhibiting the expression of _MagT1_ or _TRPM7_ or culturing the cells under Mg deficient conditions. RESULTS TRPM7 AND MAGT1 ARE

OVEREXPRESSED IN HMSC INDUCED TO DIFFERENTIATE INTO OSTEOBLASTS Confluent cells were cultured for 3, 6, 10 and 14 days in an osteogenic medium containing vitamin D (OM) or in their culture

medium (CM) as a control. By real-time PCR we demonstrate an overexpression of _TRPM7_ and _MagT1_ in cells exposed to OM for 6 and 10 days from the beginning of the experiment (Fig. 1A).

Western blot shows that both TRPM7 and MagT1 are upregulated in hMSC exposed to the osteogenic medium for 6 and 14 days (Figs 1B and S1A,B). It is noteworthy that while the expression of

_TRPM7_ and _MagT1_ drops at day 14, the protein levels remain elevated until the end of the experiment. SIRNAS AGAINST _TRPM7_ OR _MAGT1_ BOOST THE EXPRESSION OF OSTEOGENIC DIFFERENTIATION

MARKERS To investigate the role of _TRPM7_ and _MagT1_ in the osteogenic differentiation of hMSC, we transiently transfected the cells with specific siRNAs for _TRPM7_, _MagT1_ or with a

non-silencing siRNA as a control (−) and then cultured hMSC in CM for 3 days. By real-time PCR _MagT1_ expression resulted completely suppressed and _TRPM7_ expression appeared dramatically

reduced but not totally abrogated (Fig. 2A). Western blot shows the downregulation of the two proteins (Figs 2B and S2A,B). To get an overview about Mg homeostasis, we measured total

intracellular Mg 48 and 72 h after transfection without finding any significant difference between cells silencing _TRPM7_ or _MagT1_ and relative controls (Fig. 2C). In siRNA transfected

cells, we analyzed the expression of _RUNX2_ and _COL1A1_ and found it slightly upregulated even in the absence of the osteogenic cocktail (Fig. 3A). Moreover, cells downregulating _TRPM7_

or _MagT1_ maintained their sensitivity to the stimulatory effect of the osteogenic cocktail and upregulated both _RUNX2_ and _COL1A1_ more than controls (Fig. 3A). By ELISA, we demonstrate

the significant increase of RUNX2 in hMSC downregulating _TRPM7_ or _MagT1_ after 5 days of osteogenic induction (Fig. 3B), while collagen type 1 is not induced probably because its

accumulation represents a late event in osteoblastogenesis15. Osteogenic differentiation of hMSC ultimately leads to the deposition of calcium in the extracellular matrix. We therefore

evaluated calcium deposition by Alizarin Red S staining16 in silenced cells cultured in OM or CM for 14 days. In support to the results obtained by RT-PCR and ELISA, we detected some calcium

deposits in siRNA transfected cells cultured in CM, but not in controls transfected with non silencing siRNAs (Fig. 4). Upon exposure to OM, siRNA transfected hMSC deposited much higher

amounts of calcium phosphate crystals than non silenced cells. AUTOPHAGY IS INVOLVED IN ACCELERATING OSTEOGENESIS IN HMSC EXPOSED TO SIRNAS AGAINST _TRPM7_ OR _MAGT1_ Autophagy contributes

to the osteogenic differentiation of hMSC20. In agreement with these findings, we show the conversion of microtubule-associated protein 1A/1B light-chain phosphatidylethanolamine conjugate

(LC3B) to autophagosome-associated LC3B-II, which is the most widely used autophagosome marker21, in hMSC treated with siRNAs targeting _MagT1_ or _TRPM7_ for 3 days (Figs 5A and S5A,B). We

also evaluated the total amounts of beclin 1, which contributes to the initiation of autophagosome formation by interacting with phosphatidylinositol 3-kinase22. Figures 5A and S5A,B show

that beclin 1 is markedly induced in cells silencing _TRPM7_ or _MagT1_. To reinforce these results, we performed a Tandem fluorescent-tagged LC3 assay to assess autolysosome function. hMSC

were transfected with the plasmid containing the sequence for the fusion protein LC3-GFP-RFP. Briefly, in the lysosome where the pH is low, the fluorescence of GFP is quenched, while that of

RFP is stable. Formation of autophagosomes increases the number of GFP-positive/RFP-positive (yellow) vesicles, which become GFP-negative/RFP-positive (red) once fusion with lysosomes

occurs. Figure 5B confirms the induction of autophagy in cells silencing _TRPM7_ or _MagT1_ as indicated by the increase of the number of vesicles and of autophagolysosomes in silenced cells

 _vs_ controls. Considering the complex role of free calcium in autophagy23, it is noteworthy that we did not detect any modulation of intracellular free Ca in silenced cells vs their

controls (Fig. 5C). Next we tested whether bafilomycin A1, which inhibits autophagy by preventing the acidification of endosomes and lysosomes, affects the expression of _RUNX2_ in hMSC

exposed to siRNA targeting _MagT1_ or _TRPM7_ for 3 days. Figure 5D shows that bafilomycin A1 prevented the increase of _RUN_X2 expression in siRNA treated cells, thus indicating that

autophagy contributes to accelerating osteogenic differentiation in cells silencing _TRPM7_ or _MagT1_. LOW EXTRACELLULAR MG ENHANCES OSTEOGENIC DIFFERENTIATION OF HMSC BY ACTIVATING

AUTOPHAGY We asked whether Mg deprivation has an effect on hMSC differentiation and found that culture in 0.1 mM Mg for 14 days enhances Ca deposition in the extracellular matrix as detected

by Alizarin Red S staining (Fig. 6A). Accordingly, the expression of _RUN_X2 is higher in Mg deficient cells versus their controls both in the presence and in the absence of osteogenic

stimuli (Fig. 6B). No differences were observed in the content of total intracellular Mg in hMSC maintained in Mg deficient medium vs controls (Fig. 6C). Moreover, western blot shows that

TRPM7 is upregulated after 3 days and MagT1 after 6 days of culture in medium containing 0.1 mM Mg (Figs 6D and S6A,B). Next, we demonstrate that culture in low extracellular Mg (0.1 mM Mg)

stimulates the conversion of LC3B to LC3B-II and upregulates beclin 1 in our experimental model (Figs 7A and S7A,B). These results were confirmed by Tandem fluorescent-tagged LC3 assay

described above which shows an increase of the number of vesicles and of autophagolysosomes in Mg deficient hMSC (Fig. 7B). Figure 7C demonstrates no variation of intracellular free Ca

between cells cultured in 0.1 or 1.0 mM Mg medium. The contribution of autophagy to osteogenic differentiation is supported by the fact that bafilomycin A1 reduced the expression of _RUN_X2

in cells cultured in low (0.1 mM) or normal (1.0 mM) extracellular Mg for 3 days (Fig. 6B). To the best of our knowledge this is the first evidence of a link between Mg, its transporters and

autophagy. DISCUSSION Mg is required for every biological process not only because it is necessary for the function of hundreds of enzymes but also because it maintains the active

conformation of macromolecules, regulates second messengers, various transporters and ion channels12. Therefore, it is not surprising that the amounts of intracellular Mg are balanced by a

coordinated interplay among channels and transporters mediating Mg influx, exchangers regulating its efflux, and Mg shuffling from organelles to cytosol and viceversa24. About 60% of total

Mg is stored in the bone and a tight control of magnesium homeostasis is crucial for bone health13. A key event in bone formation is the differentiation of MSC into osteoblasts, a process

which involves Mg and its transporters5,6,7,11,25. It has recently been reported that TRPM7, which is important in skeletogenesis26, acts as a mechanotransducer in murine MSC and commits

their fate towards osteogenic differentiation in response to mechanical stimulation5. Also MagT111 and the Na/Mg exchanger SLC41A27 contribute to osteoblastogenesis28. We focused our studies

on TRPM7 and MagT1, both contributing to Mg transport into the cells12, in a model of hMSC induced to differentiate into osteoblasts. We show the overexpression of _TRPM7_ and _MagT1_ with

a peak after 6 days of osteogenic induction and a gradual return to control levels within 14 days. The increase of the transcripts is paralleled by the upregulation of TRPM7 and MagT1 at the

protein level, which is retained up to day 14 from osteogenic induction. We hypothesize that post-translational mechanisms might be implicated in maintaining high the amounts of TRPM7 and

MagT1 until hMSC reach full differentiation. While further studies are required, it is noteworthy that our results with MagT1 are in agreement with a report showing the increase of MagT1 in

rat MSC exposed to an osteogenic medium11. To unravel the function of TRPM7 and MagT1 in hMSC, we utilized specific siRNAs and demonstrate that downregulating _TRPM7_ or _MagT1_ accelerates

hMSC osteogenic differentiation. Our observation in hMSC silencing _MagT1_ is in disagreement with previous results in rat MSC showing that knocking down _MagT1_ inhibits osteogenic

differentiation11. This discordance could be ascribed either to the different behavior of cells of different species or to the different protocols used to induce differentiation. While we

used vitamin D to induce osteogenic differentiation, Zheng _et al_. used dexamethasone which has been shown to modulate TRPM7 in some tissues29. Since i) MagT1 and TRPM7 are upregulated in

hMSC differentiation, and ii) siRNAs targeting _TRPM7_ or _MagT1_ hasten the expression of osteogenic markers as well as Ca deposition, we hypothesize that these transporters contribute to

coordinate hMSC response to osteogenic stimuli by preventing excessive osteoblastogenesis. In this light, TRPM7 and MagT1 might be considered as sensors-controllers of the extent of hMSC

osteogenic differentiation. Silencing _TRPM7_ in different cell types, among which colon carcinoma cells, thymocytes and human endothelial cells, does not influence intracellular Mg

content30,31,32, while knocking down _MagT1_ in mammalian cell lines lowered the levels of intracellular Mg33. We did not detect differences in intracellular Mg content in hMSC silencing

_TRPM7_ or _MagT1_, suggesting that either TRPM7 or MagT1 is sufficient to maintain intracellular Mg homeostasis in hMSC. Considering that many different Mg transporters have been described

and account for Mg balance24, it is feasible to propose that also other proteins contribute to maintain Mg intracellular concentrations. A connection between bone health and autophagy has

been described17,18,19. It is noteworthy that autophagy is activated in the early phases of hMSC differentiation20. It is also interesting that resting quiescent hMSC accumulate

autophagosomes34, which indicates that the cells are ready to react to different stimuli by activating autophagy. Moreover, autophagy is induced in embryonic stem cells early during

differentiation35 and in somatic cells reprogrammed to induced pluripotent stem cells36. We suggest that siRNA targeting _TRPM7_ or _MagT1_ boosts the expression of markers of osteogenic

differentiation, at least in part, by activating autophagy, which removes unnecessary organelles, generates substrates to supply energy and recycles macromolecular blocks to drive cell

differentiation. Accordingly, bafilomycin A1, an inhibitor of autophagy, prevents the acceleration of osteogenic differentiation in our experimental model. Ca does not seem to be implicated

in triggering autophagy in cells silencing _TRPM7_ or _MagT1_ as well as in hMSC cultured in low extracellular Mg. To this purpose, it is noteworthy that early after exposure to a Mg

deficient medium, TRPM7 increases, while later, when TRPM7 declines to baseline levels, MagT1 is upregulated. We hypothesize that the sequential upregulation of the two proteins allows to

maintain unaltered the concentrations of intracellular Mg. Moreover, Mg deficiency mimics the effects of silencing _TRPM7_ or _MagT1_ because it accelerates the osteogenic differentiation of

hMSC25 and this happens in association with the trigger of autophagy without any detectable increase of free Ca. Apart from transporting Mg, TRPM7 and MagT1 serve other functions. MagT1

contributes to the oligosaccharyltransferase complex, thus being involved in the glycosylation of various proteins37. It should be noted that the components of the extracellular matrix are

glycosylated and this is important for bone formation and remodeling38. Moreover, it has been proposed that MagT1 might regulate Mg transport by glycosylating Mg transporters24. TRPM7

possesses a kinase domain and we cannot rule out the possibility that silencing _TRPM7_ might affect the phosphorylation of specific substrates, some of which have been identified24. Of

interest, one of the protein phosphorylated by TRPM7 is annexin 1, a Ca-regulated phospholipid binding protein implicated in the regulation of cell growth, apoptosis39,40 and also in

osteogenic differentiation41. On these bases, downregulating _TRPM7_ and/or _MagT1_ might impact on the post-translational modification of proteins. In conclusion, we demonstrate that MagT1

and TRPM7 as well as Mg deficiency contribute to the regulation of hMSC osteogenic differentiation partly by modulating autophagy in a Ca-independent fashion. METHODS CULTURE OF HMSC hMSC

were isolated from adult human bone marrow withdrawn from bilateral punctures of the posterior iliac crests of healthy male volunteers, after obtaining informed consent from all the subjects

at the Policlinico in Milano, in compliance with the Helsinki declaration, according to institutional guidelines and regulations of the Ethical Committee of “IRCCS Policlinico” Milano.

These cells were used in previous studies16,25. The cells were cultured at 37 °C and 5% CO2 in Dulbecco’s Modified Eagle’s Medium containing 10% fetal bovine serum and 2 mM glutamine

(culture medium, CM). All the reagents for cell culture were from Sigma-Aldrich. The cells were used between passage 2 and 6. To obtain a transient downregulation of MagT1 and TRPM7, we

utilized the stealth siRNAs developed by Qiagen for _TRPM7_ and Invitrogen for _MagT1_. siRNAs targeting _TRPM7_ were transfected using HiPerFect Transfection Reagent (Qiagen) while for

siRNAs targeting MagT1 we used Lipofectamine RNAiMAX (Thermo Fisher Scientific). Non-silencing, scrambled sequences were used as controls (−)42. Viable cells were counted using a cell

counter (Logos Biosystems). In some experiments, hMSC were cultured in Mg-free MEM (Invitrogen, Thermo Fisher Scientific) or Mg-free MEM supplemented with MgSO4 (Sigma-Aldrich) to reach the

physiological concentration, i.e. 1 mM. To induce osteogenic differentiation, hMSC were seeded in 6-well or 96-well plates. Once the cells were confluent, an osteogenic induction cocktail

was added to the medium (osteogenic medium, OM). The osteogenic cocktail contains 2 × 10−8 M 1α,25-Dihydroxyvitamin D3, 10 mM β-glycerolphosphate and 0.05 mM ascorbic acid (Sigma-Aldrich)16.

To analyze Ca deposition by hMSC, the cells were rinsed with PBS, fixed (70% ethanol, 1 h) and stained for 10 min with 2% Alizarin Red S (pH 4.2, Sigma-Aldrich)43. Alizarin Red S staining

was released from the cell matrix by incubation in 10% cetylpyridinium chloride in 10 mM sodium phosphate (pH 7.0), for 15 min and the absorbance measured at 550 nm. The experiment was

repeated three times in triplicate. Photographs were taken at 10X magnification. REAL-TIME PCR Total RNA was extracted by the PureLink RNA Mini kit (Thermo Fisher Scientific).

Single-stranded cDNA was synthesized from 0.2 µg RNA in a 20 µL final volume using High Capacity cDNA Reverse Transcription Kit, with RNase inhibitor (Thermo Fisher Scientific) according to

the manufacturer’s instructions. Real-time PCR was performed using the 7500 FAST Real Time PCR System instrument using TaqMan Gene Expression Assays (Life Technologies, Thermo Fisher

Scientific): Hs00231692_m1 (_RUNX2_), Hs00164004_m1 (_COL1A1_), Hs00918928_g1 (_TRPM7_), Hs00997540_m1 (_MagT1_). The housekeeping gene _GAPDH_ (Hs99999905_m1) was used as an internal

reference gene. Relative changes in gene expression were analyzed by the 2−ΔΔCt method16. The experiments were repeated three times in triplicate. WESTERN BLOT ANALYSIS Western blot was

performed on hMSC induced to differentiate into osteoblasts, exposed to siRNAs targeting _TRPM7_ and/or _MagT1_ or cultured in medium containing 0.1 mM Mg. After lysis, samples (80 µg/lane)

were separated on SDS-polyacrylamide gel, transferred to nitrocellulose sheets at 150 mA for 16 h, and probed with antibodies against TRPM7 (Bethyl), MagT1 (Abcam), LC3B, beclin 1 (Cell

Signalling Technology) and actin (Santa Cruz Biotechnology). Secondary antibodies were labelled with horseradish peroxidase (GE healthcare). The ECL Western Blotting Substrate (Thermo Fisher

Scientific) was used to detect immunoreactive proteins. The western blots were repeated at least three times and a representative blot is shown. Densitometry was performed using ImageJ

software and results are shown as the mean ± standard deviation. TANDEM FLUORESCENT-TAGGED LC3 ASSAY The Tandem fluorescent-tagged LC3 assay, which is based on the different pH stability of

two fluorescent proteins, allows to monitor autophagic flux and provides information about the number of vesicles and/or the progression to the late phase of autophagy44. Briefly, hMSC were

transfected with a plasmid containing the sequence coding for the LC3 protein fused with two fluorophores, specifically RFP (red) and GFP (green) using lipofectamine 2000 for 4 h.

Twenty-four hours post transfection hMSC were silenced for _TRPM7_ or _MagT1_ as described above. 48 h later the cells were fixed and analysed by confocal microscopy (LEICA SP8,

magnification 40x). The early phase, characterized by phagosome formation, is detectable through the visualization of vesicles containing the two fluorophores (yellow fluorescence). The late

phase, characterized by the fusion of the phagosome with the lysosome and the formation of the phagolysosome, is characterized by the cleavage of the GFP fragment, which is sensitive to the

acidic pH of the lysosome, so that only RFP signal is detected (red fluorescence). QUANTIFICATION OF INTRACELLULAR MG AND CA Total Mg content was measured on sonicated hMSC using the

fluorescent chemosensor DCHQ5 (kindly donated by Prof. S. Iotti, Università di Bologna) as described31,32. Fluorescence intensities were acquired at 510 nm. Mg concentrations of the samples

were obtained by the interpolation of their fluorescence with the standard curve performed using MgSO4. To quantify free Ca, Fura-2/AM (10 μM) was added to the culture medium for 60 min.

Then the cells were washed with a buffer containing NaCl 125 mM, KCl 5 mM, MgSO4 1.2 mM, CaCl2 2 mM, glucose 6 mM, Hepes-NaOH buffer 25 mM (pH 7.4), removed by trypsinization, and suspended

in the aforementioned buffer. Fluorescence intensities were acquired using a spectrophotometer with excitation wavelengths of 360 nm and emission at 450 nm45. Results were normalized on cell

number. ELISA hMSC were exposed to CM or OM for 5 days. For the quantitative determination of RUNX2 and collagen type 1, Cusabio ELISA kit were used according to the manufacturer’s

instructions. ELISAs were performed at least three times, and each sample was measured in triplicate. Data are shown as the mean ± standard deviation. STATISTICAL ANALYSIS Statistical

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12–20 (2012). CAS  PubMed  Google Scholar  Download references ACKNOWLEDGEMENTS We thank Dr. Silvia Zecchini for her help in performing the Tandem fluorescent-tagged LC3 assay and Prof.

Stefano Iotti and the Bologna team for providing the fluorescent chemosensor DCHQ5. This work was supported by intramural funds. AUTHOR INFORMATION AUTHORS AND AFFILIATIONS * Dipartimento di

Scienze Biomediche e Cliniche L. Sacco, Università di Milano, Milano, I-20157, Italy Sara Castiglioni, Valentina Romeo, Laura Locatelli, Alessandra Cazzaniga & Jeanette A. M. Maier

Authors * Sara Castiglioni View author publications You can also search for this author inPubMed Google Scholar * Valentina Romeo View author publications You can also search for this author

inPubMed Google Scholar * Laura Locatelli View author publications You can also search for this author inPubMed Google Scholar * Alessandra Cazzaniga View author publications You can also

search for this author inPubMed Google Scholar * Jeanette A. M. Maier View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS S.C. and J.A.M.

conceived and designed the experiments. S.C., V.R., L.L. and A.C. performed the experiments. S.C., V.R., L.L., A.C. and J.A.M. analyzed the data. J.A.M. wrote the paper. All authors reviewed

the manuscript. CORRESPONDING AUTHOR Correspondence to Jeanette A. M. Maier. ETHICS DECLARATIONS COMPETING INTERESTS The authors declare no competing interests. ADDITIONAL INFORMATION

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ARTICLE Castiglioni, S., Romeo, V., Locatelli, L. _et al._ TRPM7 and MagT1 in the osteogenic differentiation of human mesenchymal stem cells in vitro. _Sci Rep_ 8, 16195 (2018).

https://doi.org/10.1038/s41598-018-34324-8 Download citation * Received: 06 March 2018 * Accepted: 12 October 2018 * Published: 01 November 2018 * DOI:

https://doi.org/10.1038/s41598-018-34324-8 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not

currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * Transient Receptor Potential Melastatin (TRPM7) *

Osteogenic Differentiation * Autophagy * Magnesium Deficiency States * TRPM7 Expression