We established an LNA/DNA nanobiosensor for dynamic gene expression analysis in single hMSC during osteogenic differentiation (Fig. 1A). The LNA/DNA nanobiosensor is a double-stranded LNA donor - quencher complex (Fig. 1A). The LNA donor probe is a 20-base oligonucleotides sequence with alternating LNA/DNA monomers. The LNA nucleotides are modified DNA nucleotides with higher thermal stability and specificity . The LNA donor sequence is designed to be complementary to the target mRNA sequence. This LNA donor probe will spontaneously bind to the quencher sequence to form a LNA donor-quencher complex. The fluorophore at the 5’ end of the LNA donor is quenched by quencher sequence (Iowa Black Dark Quencher) due to its quenching ability . The LNA donor-quencher complex is then transfected into hMSCs following manufacturers’ instructions. This LNA probe is designed to have a higher binding affinity with the target sequence. Once transfected into cells, in the presence of a target mRNA, the LNA probe is thermodynamically displaced from the quencher, to bind to specific target sequences. This displacement is due to larger difference in binding free energy between LNA probe to target mRNA versus LNA probe to quencher sequence. This displacement reaction permits the fluorophore to fluorescence, thus detecting the gene expression at single cell level (Fig. 1B-C).
In order to optimize the LNA/DNA nanobiosensor for monitoring gene expression dynamics during osteogenic differentiation in both 2D and 3D microenvironments, we first characterized the optimal quencher-to-donor ratio to minimize the background noise caused by free fluorophore during the reaction. The donor concentration was set to 100 nM. The quencher concentrations were adjusted such that the quencher-to-donor ratio ranges from 0.5 to 10. The fluorescence intensity was then measured at different quencher-to-donor ratios. As shown in Fig. 2A, the fluorescence intensity decreases as the quencher-to-donor ratio increases. To acquire the optimal quencher-to-donor ratio to minimize background noise, we further calculated the quenching efficiency at different ratios. Quenching efficiency was determined by subtracting the fluorescence intensity of free fluorophore by the fluorescence intensity of the donor-quencher complex, divided by the fluorescence intensity of free fluorophore, multiplying the result by 100. Thus, quenching efficiency were calculated as 28.7%, 57.7%, 87.8%, 97.1%, 97.6%, 98.1%, 98.3%, and 98.6%, as the quencher-to-donor ratios were 0.5, 1, 1.5, 2, 4, 6, 8, and 10, respectively. This result indicates that the quenching efficiency increases as quencher-to-donor ratio increases. The intensity of fluorophore was quenched to a very low level, about 3% of the maximum intensity level, at a quencher-to-donor ratio of 2. Further increase of the quencher concentrations did not significantly reduce the fluorescence intensity, consisting to previously reported results [26, 27]. Thus, we set the quencher-to-donor ratio to 2 for the subsequent studies. We next characterized the detectable range of target concentrations of this LNA/DNA nanobiosensor. The fluorescence intensity was measured by varying the DNA target oligonucleotide concentrations while setting the LNA probe concentration at 100 nM. As shown in Fig. 2B, the sigmoid-shaped titration curve shows a large dynamic range for quantifying target concentrations ranging from 1 nM to 1000 nM, indicating this LNA probe provides a sufficient large dynamic range for detecting target mRNAs. We further examined the background fluorescence intensity of this LNA probe with different culture mediums and did not observe non-specific binding in different conditions including hMSCs culture and induction medium, Fig. 2C. Moreover, we evaluated the stability of this LNA/DNA nanobiosensor by incubating this probe with target mRNA for different duration, ranging from 1 to 14 days. As shown in Fig. 2D, the fluorescence intensity did not show a significant difference as the incubation time increased, indicating that the stability of this LNA probe is not affected by incubation time. All these results suggest that the fluorescence of this LNA probe is specific, sensitive, and stable for target mRNA detection.
Osteogenic differentiation is a dynamic cellular process that consists of distinct sub-stages and dynamic transcriptional responses during hMSCs differentiation into osteogenic or adipogenic lineages . It has been reported that there are three distinct early stages of cell fate determination during osteogenic differentiation. These three stages can be identified and determined by transcription factor dynamics when cells adopt a committed phenotype: initiate differentiation (0–3 hr, phase I), acquire lineage (4–24 hr, phase II), and early lineage progression (48–96 hr, phase III). Although previous gene expression studies have identified several critical regulatory signaling pathways that are involved in hMSCs early osteogenic differentiation, it is obscure how the gene expression dynamics relate to early osteogenic different progression. To examine the early osteogenic differentiation process, we monitored the transcription factor Dll4 mRNA expression dynamics using the LNA/DNA nanobiosensor. A random probe was included as a control, Tab. S1. Both random and Dll4 LNA probes were transfected one day before osteogenic induction. The gene expression profile was determined by measuring fluorescence intensity for each cell daily for 7 days and calculated using NIH ImageJ software. We first confirmed random probe has minimum fluorescence background in hMSCs that were cultured in both basil and osteogenic differentiation medium, Fig. 3A, Fig. S1. There is no significant difference in fluorescence intensity of hMSCs between the control group and osteogenic differentiated group, and among each group, Fig. 3C. Compared to the random probe, an increase in Dll4 mRNA expression was observed for all the cells in the osteogenic induction group. Figure 3B and Fig. S2 showed the representative bright and fluorescence images of hMSCs after 3, 5, and 7 days of osteogenic induction. The yellow arrows indicate expressed Dll4 mRNA in osteogenic differentiated hMSCs. We first observed that the Dll4 mRNA expression level was increased in osteogenic induced hMSCs compared to hMSCs cultured in basil medium. This finding is consistent with previous results reported by other groups . To further compare the Dll4 mRNA expression levels, we quantified Dll4 mRNA expression dynamics by measuring the mean fluorescence intensity of each hMSC. Interestingly, after osteogenic induction, the Dll4 mRNA expression was increased and reached a peak after 5 days of induction, then the Dll4 expression was observed to decrease and stayed at a stable level after 7 days of induction, Fig. 3D. Dll4 mRNA expression level was increased 4 folds from day 1 to day 5. After 5 days of osteogenic induction, the Dll4 mRNA expression level was reduced one-fold and kept at the same level for 6 and 7 days of induction. These results indicate that Dll4 mRNA expression dynamics indicate two different stages during osteogenic differentiation: early differentiation stage (day 1- day 5) and calcium mineralization stage (after day 5). It has been reported that the process of hMSCs osteogenic differentiation includes early lineage progression (proliferation), early differentiation, and later stage differentiation (calcium mineralization) [28, 30–32]. Our results indicate that Dll4 mRNA expression could potentially be the molecular signature of early osteogenic differentiation.
The above results showed that Dll4 mRNA expression might relate to hMSCs osteogenic differentiation. Thus, we hypothesize that Dll4 mRNA is a molecular signature of osteogenic differentiated hMSCs. To test our hypothesis, we simultaneously detected Dll4 mRNA expression and alkaline phosphatase (ALP, a biochemical marker for bone formation) enzyme activity during hMSCs osteogenic differentiation using LNA/DNA nanobiosensor and ALP staining assay (Sigma Aldrich), Fig. 4. We performed the dsLNA probe assay and immunostaining to correlate differentiated cell characteristics with the expression of Dll4 mRNA. A random probe was utilized as a negative control. The Dll4 mRNA expression and ALP enzyme activities were imaged and quantified after 5 and 10 days of osteogenic induction, respectively. Figure 4A showed representative images of hMSCs after 5 days of culture in basal medium (upper panel) and osteogenic induction medium (lower panel), respectively. Compared to undifferentiated cells, differentiated cells showed enhanced ALP activity and high expression of Dll4 mRNA. In contrast, undifferentiated cells in the control group and induction group showed low levels of Dll4 mRNA expression and ALP activities. Differentiated cells showed high levels of ALP activity, while with the random probe, there is no significant signal detected in hMSCs, Fig. S3. We next analyzed ALP activity in hMSCs in both control and induction groups. Figure 4B showed the comparison of ALP activity after 5 days and 10 days of culture, respectively. Compared to hMSCs cultured with basal medium, ALP activity in hMSCs cultured in osteogenic induction medium increased 3.2 folds and 6.5 folds after 5 and 10 days of induction, respectively. We further measured and quantified Dll4 mRNA expression of hMSCs after 5 days and 7 days of osteogenic induction. Dll4 mRNA expression levels were increased by 5.2 folds and 4.56 folds after 5 days and 10 days of osteogenic induction, compared to the control group, Fig. 4C. These results showed that Dll4 mRNA is correlated to ALP activity, which is a molecular biomarker of differentiated cells. Moreover, Dll4 mRNA expression showed a dynamic profile during osteogenic differentiation. Meanwhile, the intensity of the random probe maintained a low level regardless of differentiated or undifferentiated cells. Taken together, these results indicate that Dll4 is a molecular signature of osteogenic differentiation of hMSCs.
In order to investigate the involvement of Notch1-Dll4 signaling during hMSCs osteogenic differentiation, we perturbed Notch1-Dll4 signaling using two pharmacological drugs, DAPT and Jag1 peptide. DAPT is a γ-secretase inhibitor that blocks Notch endoproteolysis and thus serves as a Notch signaling inhibitor . Jag1 peptide can activate Notch signaling by inhibiting the function of endogenous Jag1, a Notch ligand that has weak signaling capacity but competes with Dll4 . hMSCs were treated with DAPT (20 µM) and Jag1 (40 µM) during osteogenic differentiation to examine the drug effects. A control group was designed without osteogenic induction. The osteogenic differentiation and Dll4 mRNA expression under different treatments were evaluated and compared. Osteogenic differentiation after induction was evaluated by quantifying ALP activity under different treatments. Our results showed pharmacological treatments with DAPT and Jag1 effectively induced changes in osteogenic differentiation, Fig. 5A. Specifically, DAPT treatment inhibited osteogenic differentiation and decreased Dll4 mRNA expression, Fig. 5A. After 5 days of osteogenic induction, ALP activity in the DAPT treatment group was mediated compared to the induction group without treatment. Particularly, the ALP enzyme activity decreased by 29.9% ((ALP fluorescence intensity in induction group – ALP fluorescence intensity in the presence of DAPT) / ALP fluorescence intensity in induction group) compared to the osteogenic induction group, Fig. 5C. These results indicate that inhibition of Notch1-Dll4 signaling using γ-secretase inhibitor mediates osteogenic differentiation of hMSCs. Interestingly, with the treatment of Jag1, osteogenic differentiation efficiency did not show a significant difference, Fig. 5C. However, it has been reported that Notch1 ligand, Jag1 has been demonstrated to be essential in various development processes, including osteogenesis. Deng et al. recently reported that immobilized Jag1 mimetic ligand activates Notch signaling via the upregulation of NICD, leading to the enhanced stem cell osteogenesis. Jag1 mediated Notch signaling activation is essential for stem cell proliferation and differentiation [36, 37]. A previous study showed that exposure of mouse MSC to Jag1 inhibited osteogenesis, indicating the complex role of Jag1 during stem cell differentiation .
To further investigate the effects of Notch1-Dll4 signaling during hMSCs induced osteogenic differentiation, we examined Notch1 ligand, Dll4 mRNA expression under different treatments using LNA/DNA probe. Compared to the control group, hMSCs cultured with the osteogenic induction medium showed a significant increase (~ 4 folds) in the expression of Dll4 mRNA. Meanwhile, with the treatment of γ-secretase inhibitor DAPT, a significant decrease of Dll4 mRNA (32.7%) was observed compared to the osteogenic induction group, Fig. 5B. Jag1 treatment also reduced the Dll4 mRNA expression compared to the induction group. However, compared to the DAPT group, Dll4 expression was increased with the treatment of Jag1. These results suggest a positive correlation between Dll4 mRNA expression and osteogenic differentiation. As the Notch pathway is triggered by binding specific ligands to receptors, our results indicate that the expression of Notch ligand Dll4 was modulated during osteogenic differentiation. Dll4 mRNA was significantly increased at the end of the differentiation period (Fig. 5B). Notch inhibitors induced a significant decrease of ALP enzyme activity and Dll4 mRNA expression compared to osteogenic induction.
Recently, there has been a growing interest in understanding hMSCs differentiation in 3D microenvironments. It has been shown that the self-assembly of hMSCs into tightly packed clusters of cells in each aggregate mimic “in vivo-like” microenvironment and preserves hMSCs phenotype and innate properties [38, 39]. Moreover, it has been reported that the formation of 3D aggregates, or spheroids enhanced the regenerative capacity of hMSCs by promoting the secretion of proangiogenic and chemotoxic factors, and improved cell retention, and survival in preclinical studies . Here, we investigated the effects of Notch1-Dll4 signaling in regulating osteogenic differentiation of 3D hMSCs spheroids. We first fabricated 3D self-organized spheroids of hMSCs using the hanging drop approach . The self-organized 3D spheroids were collected and seeded in Matrigel, Fig. S5. To reveal the mechanisms of Notch1-Dll4 signaling, 3D hMSCs spheroids were treated using DAPT and Jag1, respectively. A control group without osteogenic induction was designed for comparison. Figure 6A showed the representative images of hMSCs spheroids under different treatments. Compared to the control group, hMSCs spheroids cultured in osteogenic induction medium showed increased ALP activity and Dll4 mRNA expression, Fig. 6A-B. Both DAPT and Jag1 treatments inhibited osteogenic differentiation of 3D hMSCs spheroids. We further quantified the ALP activities and the size of spheroids under different treatments. Compared to the control group, ALP activity was increased ~ 18 folds for the spheroids in the osteogenic induction group. With Notch pathway inhibitor DAPT treatment, the expression of ALP was significantly decreased, with a 54.2% decrease compared to the induction group. Meanwhile, Jag1 treatment resulted in a 30.2% decrease compared to the induction group. We next examined the sizes of the spheroids under different treatments. Without osteogenic induction, the diameter of the 3D spheroids is approximately 266.5 µm, Fig. 6C. The diameter of the spheroids was decreased by 18.6% in the osteogenic induction group. In contrast, compared to the induction group, the diameter of the spheroids was increased by 33.3% and 31.3% with the treatments of DAPT and Jag1, respectively, Fig. 6C. These results indicated that inhibition of the Notch1-Dll4 pathway by γ-secretase inhibitor DAPT could inhibit osteogenic differentiation and enhance the proliferation of hMSCs spheroids.