Induction of human stem cells into ameloblasts by reaggregation strategy

Abstract

Background

Human epithelium-derived stem cells and induced pluripotent stem cells (hiPSCs) possess the capability to support tooth formation and differentiate into functional enamel-secreting ameloblasts, making them promising epithelial-component substitutes for future human tooth regeneration. However, current tissue recombination approaches are not only technically challenging, requiring precise induction procedures and sophisticated microsurgery, but also exhibit low success rates in achieving tooth formation and ameloblastic differentiation.

Methods

Suspended human keratinocyte stem cells (hKSCs) or cells from three hiPSC lines were directly mixed with dissociated embryonic mouse dental mesenchymal cells (mDMCs) that possess odontogenic potential in different proportions and reaggregated them to construct bioengineered tooth germs. The success rates of tooth formation and ameloblastic differentiation were confirmed after subrenal culture. The sorting capability, sequential development, and ameloblastic differentiation of stem cells were examined via GFP tracing, RT-PCR, and histological analysis, respectively.

Results

Our reaggregation approach achieved an impressive success rate of more than 90% in tooth formation and 100% in ameloblastic differentiation when the chimeric tooth germs contained 1%~10% hKSCs or 5% hiPSCs. In addition, we observed that hiPSCs, upon exposure to mDMCs, initially transformed into epidermal cells, as indicated by KRT14 and CD29 expression, before progressing into dental epithelial cells, as indicated by SP6 and SHH expression. We also found that epithelial-derived hiPSCs, when reaggregated with mDMCs, were more favorable for tooth formation than their mesenchymal-derived counterparts.

Conclusions

This study establishes a simplified yet highly effective cell-cell reaggregation strategy for inducing stem cells to support tooth formation and differentiate into functional ameloblasts, paving the way for novel approaches for the development of stem cell-based tooth organoids and bioengineered tooth germs in vitro.

Supplementary Information

The online version contains supplementary material available at 10.1186/s13287-024-03948-1.

Background

The loss of teeth, whether due to dental caries, periodontal disease, or genetic disorders, is a widespread problem that has significant impacts on oral health and overall well-being. Current treatment options are often limited and do not fully restore the function of natural teeth. Therefore, there is a critical need to develop novel approaches for tooth regeneration. Stem cell research offers great potential for addressing this challenge [1]. Studies have demonstrated that reaggregation of dissociated embryonic dental epithelial and mesenchymal cells is capable of forming bioengineered tooth germs and developing into teeth after subrenal culture. These reaggregated cell constructs were transplanted into the kidney capsule for subrenal culture, where they undergo further development into teeth. Importantly, transplantation of these bio-tooth germs into adult alveolar sockets can form fully functional regenerated teeth with physiological properties [1–3]. These studies highlight the promise of stem cell-based whole-tooth regeneration as a therapeutic strategy for tooth replacement [1, 4–6].

Given that tooth development requires reciprocal and sequential interactions between dental epithelium and mesenchyme, stem cell-based whole-tooth regeneration also requires the interaction of dental epithelial-derived and mesenchymal-derived cells [1, 5–7]. Compared to postnatal human dental mesenchymal stem cells, postnatal human dental epithelial-derived stem cells are absent due to ameloblastic apoptosis during tooth eruption. Human non-dental epithelial-derived postnatal stem cells, such as hKSCs [8–10] and gingival epithelial (HGEp) cells [11], as well as hiPSCs [12], have been shown to possess the capability to support tooth formation and differentiate into functional ameloblasts. However, the tissue recombination approaches are technically demanding and exhibit low success rates.

Here, we developed a simplified yet effective method for inducing human postnatal stem cells to support tooth formation and differentiate into ameloblasts. We directly mixed suspended hKSCs or hiPSCs with dissociated embryonic mouse dental mesenchymal cells (mDMCs) that possess odontogenic potential and reaggregated them to construct bioengineered tooth germs. This cell-cell reaggregation strategy eliminates the need for prior induction of stem cells into epithelial sheets, the addition of growth factors, and complex micro-recombination techniques, achieving a success rate of more than 90% in tooth formation and 100% in ameloblastic differentiation when the chimeric tooth germs contained 1%~10% hKSCs or 5% hiPSC.

Methods

Cell culture

hKSCs derived from pediatric foreskin tissue were exposed to lentivirus-GFP for 24 h. The third generation of hKSCs, which reached approximately 90% confluence, was utilized in the reaggregation [9, 10]. Three hiPSC lines, Vu1-hiPSCs, A4244-hiPSCs, and XVF-hiPSCs, derived from urine cells, peripheral blood monocytes, and foreskin fibroblasts, respectively, were obtained from the laboratory of Pei at the Guangzhou Institute of Biomedicine and Health, Chinese Academy of Sciences [12]. These hiPSC lines were cultivated in mTeSR^TM1 medium (StemCell Technologies) and assessed for multipotency markers, including OCT-4 (ABCam, ab19857) and SSEA-4 (ABCam, ab16287) in Appendix Fig. 1.

Reaggregation, organ and subrenal culture

Pregnant mice were sacrificed in a CO₂ chamber. To make a single-cell suspension, E13.5 molar mesenchyme was dissected using a pair of sharpen forceps under a stereo microscope from wild-type mandibles (the C57/B6 background, Shanghai SLAC Laboratory Animal CO. LTD, China), where the epithelium had been removed after enzyme digestion, or from the mandibles of GFP transgenic line [13]. The isolated dental mesenchymal tissues were pooled in the culture medium and dispersed into a single-cell suspension with the aid of a micropipette. The suspended mDMCs were blended with hKSCs (GFP-labeled) or hiPSCs in varying proportions and reaggregated into cell pellets via centrifugation, as previously described [14, 15]. After incubation in Eppendorf tubes for 2 h, the cell pellets were transferred to Trowell-type organ culture dishes and sustained for 12 h before being subjected to subrenal culture in nude mice. Anesthesia was induced by inhalation (5% isoflurane) via a small animal anesthesia machine (RWD Life Science Co.,Ltd). Each type of reaggregated graft was performed in Table 1. The relative experiments have complied with updated ARRIVE 2.0 guidelines.

Table 1.

Success ratio of tooth formation and ameloblastic differentiation in reaggregated grafts

Proportion of cells			Number of grafts	Number of tooth formation (%)	Number of ameloblastic differentiation (%)
Stem cells		mDMCs
hKSCs	0%	100%	15	0 (0)	/
	1%	99%	16	14 (87.5)	14 (100)
	5%	95%	12	11 (91.7)	11 (100)
	10%	90%	28	26 (92.9)	26 (100)
	20%	80%	23	15 (65.2)	15 (100)
	30%	70%	29	0 (0)	/
	40%	60%	27	0 (0)	/
vU1-hiPSC	2%	98%	15	13 (86.7)	13 (100)
	5%	95%	15	14 (93.3)	14 (100)
	10%	90%	14	10 (71.4)	10 (100)
	20%	80%	18	4 (22.2)	4 (100)
XVF-hiPSC	5%	95%	19	13 (68.4)	13 (100)
	10%	90%	12	5 (41.6)	5 (100)
A4244-hiPSC	5%	95%	16	13 (81.2)	13 (100)
	10%	90%	14	8 (57.1)	8 (100)

mDMCs, mouse dental mesenchymal cells from E13.5 mandibular molar germs; hKSCs, human keratinocyte stem cells; hiPSCs, human induced pluripotent stem cells; Vu1-hiPSCs, hiPSCs derived from urine cells; A4244-hiPSCs, hiPSCs derived from peripheral blood monocytes; XVF-hiPSCs, hiPSCs derived from urine cells foreskin fibroblasts

Histology, RT-PCR, and immunostaining

The harvested samples were fixed with a 4% PFA/PBS, dehydrated, embedded in paraffin, and sectioned at a thickness of 7 μm. Ossified tissues were demineralized with EDTA/PBS for one week before dehydration. Histological analysis was conducted using standard H&E and Azan dichromic staining. Total RNA was extracted utilizing Trizol (Invitrogen), followed by reverse transcription using the Takara RT-PCR kit. Primer information is shown in Appendix Table 1, synthesized from Sangon Biotech Co (Shanghai, China). Immunostaining was carried out according to our previous protocol [8]. The primary antibodies were sourced from Abcam plc (Cambridge, UK): SSEA-4 (ab16287), OCT-4 (ab19857) and GFP (ab183734). Each experiment was performed with at least three biological replications.

Raman spectroscopy assay

Regenerated teeth were isolated from the grafts and utilized to acquire spontaneous Raman spectra using a confocal Raman spectrometer (Renishaw Invio) equipped with a 785 nm excitation laser and a 20× objective. The laser power was set at 5 mW, with an exposure time of 10 s and an accumulation count configured to 1. The spectral detection range was defined as 500 ~ 3200 cm^− 1, and a Peltier-cooled CCD with a resolution of 400 × 578 pixels was employed to collect spectral signals. The spectrometer was calibrated using the Raman line of silicon at 520 cm^− 1. For each sample, three spectra were collected from different locations within the sample to obtain a mean spectrum for use in subsequent data analysis.

Results

Efficient tooth formation and ameloblastic differentiation

In previous studies, the tissue recombination techniques have demonstrated limited success in tooth formation [9, 10, 12]. Here, we mixed hKSCs or hiPSCs with dispersed E13.5 mDMCs and immediately reaggregated to create tooth grafts, ensuring optimal cell interactions, followed by two weeks of subrenal culture, as illustrated in Fig. 1A. Histological examination revealed that hKSCs were efficiently transformed into ameloblasts in these tooth germs with remarkable efficiency of more than 90%, as evidenced by the successful formation of teeth in 51 out of 56 cases of grafted chimeric tooth germs containing 1–10% hKSCs. Furthermore, they exhibited a perfect 100% rate of ameloblastic differentiation (Table 1). However, as the proportion of hKSCs increased, there was a significant decrease in the rate of tooth formation. Actually, when the proportion of hKSCs exceeded 30%, no tooth formation was observed at all (Table 1). These findings highlight the exceptional efficiency of the cell-cell reaggregation strategy in guiding hKSCs toward differentiation to functional ameloblasts, also emphasizing that the proportion of stem cells used in this approach serves as a pivotal determinant of its success.

Fig. 1.

Spontaneous sorting of stem cells from mDMCs to form epithelial tissues. (A) Schematic diagram depicting the cell-cell reaggregation strategy. Dental mesenchymal tissues from E13.5 mice are dissociated into single-cell suspensions and mixed with hKSCs or hiPSCs in varying proportions, reaggregated with low-speed centrifugation to make cell pellets, and followed by subrenal culture. (B) Mixture of 10% GFP-labeled hKSCs and 90% mDMCs in a cell culture dish. Note the uniform distribution of GFP-labeled hKSCs shown in the fluorescence section image of the reaggregate containing hKSCs (GFP+, white arrows) and mDMCs after low-speed centrifugation (inserted). (C) The fluorescent section image of the reaggregate containing hKSCs (GFP+, white arrowheads) and mDMCs after 6 h of organ culture. (D) The whole mount and section (inserted) images of reaggregates of hKSCs (GFP+, white arrowhead) and mDMCs after 1 day of organ culture. Note the complete sorting of hKSCs from mDMCs. (E) Mixture of 5% vU1-hiPSCs (white arrows) and 95% mDMCs (GFP+), dispersed from E13.5 Rosa-Egfp transgenic mouse embryos, in a cell culture dish. (F) The fluorescence section image of the reaggregate of vU1-hiPSCs and mDMCs (GFP+) after 1 day of organ culture. Note the sorting of hiPSCs (white arrowhead) from mDMCs. (G) The whole mount image of the reaggregate of vU1-hiPSCs (white arrowhead) and mDMCs (GFP+) after 1 day of organ culture. mDMCs, mouse dental mesenchymal cells; hKSCs, human keratinocyte stem cells; hiPSCs, human induced pluripotent stem cells. Scale Bar = 100 μm (B-G)

To assess the capacity of hiPSCs in the reaggregation strategy, we used the Vu1-hiPSCs, an epithelial-derived hiPSCs previously utilized in the tooth tissue recombination [12]. We also observed a remarkable success rate of 93.3% tooth formation and 100% ameloblastic differentiation in chimeric tooth germs containing 5% hiPSCs (Table 1). A remarkable increase was also observed in chimeric tooth germs containing 5% hiPSCs from the other two hiPSC lines, A4244-hiPSCs and XVF-hiPSCs that are derived from mesenchymal somatic cells. Although the efficiency was slightly inferior to that of their epithelial-origin Vu1-hiPSCs counterpart, it still reached around 70% and 80%, respectively (Table 1). In contrast to the previously reported 28.8% efficiency in chimeric tooth germs using tissue recombination [12], our data indicate the exceptional efficiency of the cell-cell reaggregation strategy in inducing hiPSCs from different sources into functional ameloblasts. Furthermore, we observed a consistent decrease in the rate of tooth formation in all three types of chimeric tooth germs as the proportion of hiPSCs increased, consistent with the results obtained using chimeric reaggregates of hKSCs and mDMCs.

Autonomous sorting of stem cells from mDMCs to form the epithelial tissue

Previous study has shown that dental epithelial cells have the ability to sort themselves out from dental mesenchymal cells and organize into dental epithelial tissue, which supports tooth formation upon reaggregation of dissociated molar germ [14]. To compare the sorting capability of non-dental epithelium-derived hKSCs and hiPSCs when reaggregated with mDMCs with that of the dental epithelial cells, we tracked their behaviors within the reaggregated tooth germs. Despite the larger size of hKSCs compared to mDMCs, we observed a uniform distribution of GFP-labeled hKSCs in the reaggregated cell pellets after centrifugation (Fig. 1B). The majority of hKSCs had sorted out after 6 h of organ culture (Fig. 1C) and organized into epithelial structures within 24 h (Fig. 1D), which is comparable to dissociated mouse dental epithelial cells [14]. We also observed similar cell behaviors that hiPSCs sorted out from mDMCs and arranged into epithelial tissues after 24 h of organ culture in reaggregates of eGFP-labeled mDMCs and vU1-hiPSC at a proportion of 5% (Fig. 1E-G). These data demonstrate that both hKSCs and hiPSCs, when mixed with mDMCs, possess the inherent capacity to spontaneously sort out and organize into epithelial structures, paralleling the behavior of the dispersed dental epithelial cells.

Histogenesis of ameloblastic differentiation

The development of primary teeth in humans is a prolonged process that lasts approximately 400 days, beginning in the 6th week of embryogenesis and erupting in the 6th postnatal month. In contrast, mouse tooth development is much shorter, lasting only about 20 days from tooth fate determination at E10.5 to eruption at day 10 postpartum [15]. Our previous study demonstrated that this duration of tooth development is dominated by dental mesenchyme [15]. Notably, both hKSCs and hiPSCs were able to differentiate into ameloblasts and form tooth crowns within only 4 weeks of subrenal culture after recombination with mouse dental mesenchymal tissues [9, 10, 12], consistent with the duration observed in mice. To assess the developmental process in our cell-cell reaggregated chimeric tooth germs and to compare them with the previous tissue-recombined results, we performed a histological analysis and showed that the epithelial tissues arranged by hKSCs rapidly progressed to form an early dental epithelial cap within 5 days of culture (Fig. 2A). By day 7, these germs exhibited typical dental epithelial histogenesis, processing to the early bell stage (Fig. 2B). Mouse-origin odontoblasts began dentin deposition at day 9 (Fig. 2C), while hKSC-derived ameloblasts secreted enamel by day 12 (Fig. 2E), leading to the formation of well-mineralized teeth within 30 days (Fig. 2F). This developmental pace is consistent with both our previous tissue-recombination model in mice [10] and the normal development pace of mouse teeth. In the case of hiPSCs, we observed a similar developmental process, with complete tooth formation occurring within the comparable timeframe in line with the previous hiPSC tissue-recombined approach [12] and normal mouse tooth development (Fig. 3A-G). Raman spectroscopy analysis confirmed that the chemical constituents of the regenerated teeth closely resembled those of human and mouse teeth (Fig. 3H). These results provide compelling evidence for the efficient and rapid differentiation of hKSC/hiPSC into functional ameloblasts using cell-cell reaggregation strategies. However, we observed an approximately one-day delay in differentiation noted in the hiPSC-chimeras compared to hKSC-chimeras. Specifically, hiPSC-chimeras developed to the cap and bell stages after 7 and 9 days, respectively, (Fig. 3B, C), and the initiation of dentin and enamel secretion after 10 and 12 days, respectively, in subrenal culture (Fig. 3D, E).

Fig. 2.

Histogenesis of hKSC-derived dental epithelium in human-mouse chimeric teeth. (A-E) Histological staining of chimeric grafted teeth formed by the reaggregation of hKSCs (GFP+) and E13.5 mDMCs after 5 (A), 7 (B), 9 (C), 11 (D), and 12 (E) days of subrenal culture. Note the dentin deposition initiated by mouse-origin odontoblasts at day 9 and extensive enamel deposition by hKSC-derived ameloblasts at day 12. (F) Lateral view of chimeric teeth formed from hKSC-mDMC reaggregates after 30 days of subrenal culture. de, dental epithelium; dm, dental mesenchyme; am, ameloblasts; od, odontoblasts; d, dentin; e, enamel. Scale Bar = 50 μm (A-E), 500 μm (F)

Fig. 3.

Histogenesis of hiPSC-derived dental epithelium in human-mouse chimeric teeth. (A-F) Histological staining of chimeric grafted teeth formed by the reaggregation of hiPSCs and E13.5 mDMCs (GFP+) after 5 (A), 7 (B), 9 (C), 10 (D), 12 (E) and 15 (F) days of subrenal culture. Note the onset of dentin and enamel deposition by mouse-origin odontoblasts on day 10 and hiPSC-derived ameloblasts on day 12, respectively. (G) Lateral view of hiPSC-mDMC chimeric teeth formed after 30 days of subrenal culture. (H) Fluorescence section image of hiPSC-mDMC chimeric teeth after 10 days of subrenal culture. Note the absence of GFP + cells in hiPSC-derived ameloblasts within the chimeric teeth. (I) Raman spectroscopy analyses of enamel from human and mouse teeth and vU1-iPSCs derived chimeric teeth. Note great similarities among all groups in Raman peaks. de, dental epithelium; dm, dental mesenchyme; am, ameloblasts; od, odontoblasts; d, dentin; e, enamel. Scale Bar = 50 μm (A-F, H), 500 μm (G)

Sequential development of stem cells into functional ameloblasts

To investigate the differentiation path of hiPSCs into ameloblasts, we utilized epidermal markers that are not expressed in dental mesenchyme, including CD29 [16] and KRT14 (expressed in both epidermal hKSCs and human dental epithelium, but not in vU1-hiPSCs) and the dental epithelium markers SP6 and SHH (both expressed in the human dental epithelium, but not in hKSCs and vU1-hiPSCs) (Fig. 4A). We found that the initial expression of epidermal markers KRT14 after 3 days and CD29 after 4 days of culture, preceded the expression of dental epithelium markers SHH and SP6 after 7 days (Fig. 4B). These results indicate that hiPSCs undergo a sequential epithelial-to-ameloblastic transition process during the differentiation from human stem cells to epidermal cells, followed by a transition to dental epithelial cells and ultimately to ameloblasts.

Fig. 4.

Sequential expression of genes critical for dental epithelial cell transition in hiPSCs chimeric tooth germs. (A) RT-PCR detection of critical genes expressed in dental epithelial tissues, hKSCs, vU1-hiPSCs, A4244-hiPSCs, and XVF-hiPSCs, including MSX2, PITX2, BMP4, SP3, SP6, KRT14, SHH, and CD29. (B) RT-PCR analysis of critical epidermal and dental epithelial cell markers expressed in chimeric tooth germs reaggregated from hiPSCs and E13.5 mDMCs after different stages of subrenal culture. Full-length gels are presented in Appendix Figs. 2, 3, 4 and 5

To rule out the potential contamination of mouse dental epithelial cells in the chimeric teeth, we had previously performed immunostaining in the recombinant of hKSCs and mDMCs using specific antibodies against mouse or human MHC I antigens to confirmed the mouse origin of the dental pulp and the human origin of the epithelial component, respectively [9]. In the current study, we further used genetic tracing method via reaggregating hiPSCs with GFP-labeled mDMCs to exclude the contamination. Immunofluorescence staining confirmed the absence of GFP + cells in hiPSC-derived ameloblasts within the chimeric teeth (Fig. 3I). Additionally, as a control, we directly transplanted reaggregated mDMC pellets into nude mice for subrenal culture for 30 days All 15 grafted samples either formed tiny bone-like tissues or underwent degeneration (data not shown).

Discussion

Embryonic dental cells have been used to develop successful whole-tooth engineering approaches [1–3]. Built upon these seminal works, a scaffold-free stem cell-based blueprint for whole-tooth regeneration was proposed [4, 5]. It involves the induction of human postnatal epithelial-derived stem cells or hiPSCs into epithelial components and human postnatal dental mesenchymal-derived stem cells or hiPSCs into mesenchymal components harboring odontogenic potential. Following tissue recombination, a functional replacement tooth is regenerated by an in vitro organ culture of the recombinant tooth germ to the late bud or early cap stage, and subsequent implantation is made into the missing tooth site of the patient.

Although several types of dental mesenchymal stem cells isolated from adult human teeth have been shown to have the potential to regenerate tooth mesenchymal components, the identification of postnatal stem cells as the epithelial component for tooth regeneration poses significant challenges, as the postnatal dental epithelium-derived stem cells are absent due to ameloblastic apoptosis during tooth eruption. Studies have demonstrated that in the presence of tooth-inducing signals, various postnatal epithelium-derived stem cells, including HGEp cells, hKSCs, and hiPSCs [12], can be induced to differentiate into functional ameloblasts using tissue recombination strategies. These stem cells are ideal human stem cell sources for epithelial substitutes in future whole-tooth regeneration. However, they suffer from low efficiency in tooth formation rate (less than 30%) and require complex processing procedures, including sophisticated micro-recombination techniques, prior induction of stem cells into the epithelial sheet, and/or the addition of growth factors in the previous tissue-recombination strategies [9–12, 17]. Our study successfully addresses these limitations by introducing a simplified cell-cell reaggregation approach. This method involves only cell mixing and reaggregation with low-speed centrifugation, resulting in a tooth formation rate of more than 90%, with a remarkable 100% success rate in ameloblastic differentiation for both hKSCs and hiPSCs. This reaggregation approach may be valuable for regenerative dentistry and oral tissue engineering in the future.

Cell-cell communication is fundamental in developmental biology and regeneration, ensuring the biological functionality and microenvironmental hemostasis of cells, organs, and entire organisms [18]. In the context of whole-tooth regeneration, which mimics embryonic tooth development, the importance of cell-cell communication between epithelial and mesenchymal cells is particularly evident, as seen in periodontal and bone regeneration studies [19, 20]. Previous studies on whole-tooth regeneration by tissue recombination exhibited limited efficiency in tooth formation and ameloblastic differentiation [9, 10, 12, 17]. We hypothesize that, compared to normal development, stem cells may require stronger signaling to activate developmental processes in the regenerative system and that this limited efficiency may be attributed to inadequate cell-cell interaction and communication between epithelial and mesenchymal cells during the tissue recombination process. Under tissue recombination conditions, only cells located at the interface of epithelial and mesenchymal tissues are capable of reciprocal interaction and receiving inductive signals from each other, while all stem cells can interact with dental mesenchymal cells in reaggregation system, receiving adequate induction to activate the following development of tooth formation. Preceding research has demonstrated that when dissociated epithelial and mesenchymal cells from molar tooth germs are mixed and reaggregated, the epithelial cells autonomously sort from the mesenchymal cells and organize into distinct dental epithelial structures, leading to the formation of well-structured tooth organs [14, 21]. This insight offers a novel approach for constructing bioengineered tooth germs for whole-tooth regeneration by simply mixing and reaggregating epithelial and mesenchymal cells. The primary of this strategy lies in its potential to enhance the induction of epithelial cells into ameloblasts by facilitating improved cell-cell contact and communication between epithelial and mesenchymal cells. Our results corroborate the feasibility and superiority of this approach. Importantly, within this cell-cell reaggregation framework, the proportion of epithelial to mesenchymal cells emerges as a critical determinant in the rate of tooth formation and ameloblastic differentiation in bioengineered tooth germs. According to our findings, the ideal proportion of hKSCs and hiPSCs when reaggregated with mDMCs is around 5-10% and 5%, respectively (Table 1). An increase in the proportion of these stem cells leads to a marked decrease in the success rate (Table 1). This decrease is also likely caused by the inadequate inductive signaling transmitted by the dental mesenchymal cells due to the presence of the extra amount of stem cells. Therefore, in future research, an optimal proportion between stem cell types for reaggregating bio-tooth germs is crucial.

Adult stem cells, including iPSCs, retain the epigenetic memory of their original reprogrammed somatic cells [22, 23]. This memory functionally affects the behavior of stem cells and their derivatives, proving advantageous for differentiation into tissues of the donor cell lineage [24, 25]. In line with this notion, our findings reveal that epithelial-derived hiPSCs (Vu1-hiPSCs) were more favorable for tooth formation than their mesenchymal-derived counterparts (A4244-hiPSCs and XVF-hiPSCs). This highlights the importance of cell origin in determining the fate of stem cells during tooth regeneration, which provides valuable insights for the future application of hiPSCs in whole-tooth regeneration, suggesting a preference for hiPSCs reprogrammed from epithelial somatic cells for the epithelial component and those from mesenchymal somatic cells for the mesenchymal component. Additionally, we also found that the morphological phenotype of regenerated teeth in the hiPSCs group is significantly weaker than in the hKSCs group, particularly the delay in root formation and atypical tooth shape. This weakness may also be likely related to the epigenetic memory of hiPSCs as they were derived from urine cells rather than epidermal tissues. We believe that the epigenetic modification retained in the adult stem cells may play pivotal roles in regenerative tooth morphogenesis and that adult stem cells, including hiPSCs derived from epidermal tissues, such as skin [26] and the oral epithelium [27, 28], may be better cell sources for the dental epithelial component in whole tooth regeneration.

Cell adhesion and sorting are physical processes that form the basis of juxtacrine signaling and morphogenesis. The reaggregated cells undergo spatial segregation, allowing each cell type to sort to its correct embryonic position instead of mixing together. When hKSCs or hiPSCs are mixed with mDMCs in a reaggregate, the epithelium-derived stem cells migrate and aggregate to the periphery of the reaggregate, while the mDMCs migrate and adhere to the inner surface of the stem cell population. It has been shown that sorting one type of cell from another is caused by variations in cell membrane composition, typically involving calcium-dependent adhesion molecules such as cadherins. These cadherins can trigger cell sorting based on both quantitative (differing amounts of cadherins) and qualitative (various types of cadherins) distinctions [29, 30]. At this point, we may not be worried much about sorting different types of stem cells when reconstituting bio-engineered tooth germs in future regeneration applications.

The developmental pace for bioengineered tooth germs is paramount, as it is fundamental to the ultimate outcome of suitable tooth size and sufficient mineral deposition. In the developing human deciduous teeth, ameloblastic differentiation commences around the 15th week of gestation, leading to the secretion and deposition of enamel on the dentin surface by the 18th week. Six months after birth, ameloblasts undergo apoptosis during tooth eruption. Interestingly, we observed a much more rapid differentiation of hKSCs/hiPSCs into functional ameloblasts in the present and previous studies [10, 12], which are comparable to the developmental tempo in normal mouse teeth. This notion is consistent with our findings that tooth developmental tempo complies with the hereditary nature of dental mesenchyme [15]. Acquiring an in-depth understanding of this mechanism is crucial in laying the foundation for whole tooth regeneration and enhancement of upcoming therapeutic strategies if human bioengineered tooth germs could grow rapidly, mineralize, and mature to attain optimal teeth in a short time. Our previous study has suggested that mesenchymal extrinsic signals, such as FGF8, could be pivotal in controlling the lengthy developmental pace in human odontogenesis [15].

Conclusions

We have developed a streamlined yet highly effective cell-cell reaggregation method for inducing human non-dental epithelial-derived postnatal stem cells, such as hKSCs and hiPSCs, to support tooth formation and differentiate into ameloblasts. This approach achieves a remarkable success rate of over 90% in tooth formation and 100% in ameloblastic differentiation, thereby circumventing the prerequisite of pre-inducing stem cells into epithelial sheets, the necessity of growth factor supplementations, and intricated micromanipulation techniques, as previously documented. This reaggregation technique holds potential as a promising strategy for stem cell-based whole-tooth regeneration in future tooth replacement therapy. Moreover, although the human tissue-derived stem cells were tested in our system, it is based on a chimeric model with the mouse dental tissue. To testify the feasibility of this strategy and/or to determine appropriate parameters, such as the ideal proportion of epithelial and mesenchymal component cells in big animal or primate models, indeed warrants further study.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Abbreviations

hiPSCs

Human induced pluripotent stem cells

hKSCs

Human keratinocyte stem cells

mDMCs

Mouse dental mesenchymal cells

HGEp cells

Human gingival epithelial cells

GFP

Green fluorescent protein

Author contributions

CsL and XfH were responsible for conception and design. CsL, SyL, and MjH were responsible for data acquisition and analysis. CsL and YdZ were responsible for sources and data interpretation. CsL, XfH and YdZ were responsible for drafted and critically revised the manuscript. All authors read and approved the final manuscript.

Funding

This study was supported by the National Natural Science Foundation of China (82170917 and 82001002) and the Fujian Provincial Engineering Research Center of Oral Biomaterial, School and Hospital of Stomatology, Fujian Medical University (2023GC-B01 and 2022GXA01).

Data availability

My manuscript has data included as electronic supplementary material.

Declarations

Ethics approval and consent to participate

The study was reviewed and approved by the Animal Ethical and Welfare Committee of Fujian Normal University (Title: Functional mechanism of a novel BMP signaling pathway in early tooth development; Approval NO. IACUC-20200010; Approval date: April 1, 2020). hKSCs were harvested from circumcised human foreskins from children 5–12 years old whose parents gave informed consent for the study.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.