Hes1 marks peri-condensation mesenchymal cells that generate both chondrocytes and perichondrial cells in early bone development.

Bone development starts with condensations of undifferentiated mesenchymal cells that set a framework for future bones within the primordium. In the endochondral pathway, mesenchymal cells inside the condensation differentiate into chondrocytes and perichondrial cells in a SOX9-dependent mechanism. However, the identity of mesenchymal cells outside the condensation and how they participate in developing bones remain undefined. Here we show that mesenchymal cells surrounding the condensation contribute to both cartilage and perichondrium, robustly generating chondrocytes, osteoblasts, and marrow stromal cells in developing bones. Single-cell RNA-seq analysis of Prrx1-cre-marked limb bud mesenchymal cells at E11.5 reveals that Notch effector Hes1 is expressed in a mutually exclusive manner with Sox9 that is expressed in pre-cartilaginous condensations. Analysis of a Notch signaling reporter CBF1:H2B-Venus reveals that peri-condensation mesenchymal cells are active for Notch signaling. In vivo lineage-tracing analysis using Hes1-creER identifies that Hes1+ early mesenchymal cells surrounding the SOX9+ condensation at E10.5 contribute to both cartilage and perichondrium at E13.5, subsequently becoming growth plate chondrocytes, osteoblasts of trabecular and cortical bones, and marrow stromal cells in postnatal bones. In contrast, Hes1+ cells in the perichondrium at E12.5 or E14.5 do not generate chondrocytes within cartilage, contributing to osteoblasts and marrow stromal cells only through the perichondrial route. Therefore, Hes1+ peri-condensation mesenchymal cells give rise to cells of the skeletal lineage through cartilage-dependent and independent pathways, supporting the theory that early mesenchymal cells outside the condensation also play important roles in early bone development.

Bone marrow endosteal stem cells dictate active osteogenesis and aggressive tumorigenesis.

The bone marrow contains various populations of skeletal stem cells (SSCs) in the stromal compartment, which are important regulators of bone formation. It is well-described that leptin receptor (LepR)+ perivascular stromal cells provide a major source of bone-forming osteoblasts in adult and aged bone marrow. However, the identity of SSCs in young bone marrow and how they coordinate active bone formation remains unclear. Here we show that bone marrow endosteal SSCs are defined by fibroblast growth factor receptor 3 (Fgfr3) and osteoblast-chondrocyte transitional (OCT) identities with some characteristics of bone osteoblasts and chondrocytes. These Fgfr3-creER-marked endosteal stromal cells contribute to a stem cell fraction in young stages, which is later replaced by Lepr-cre-marked stromal cells in adult stages. Further, Fgfr3+ endosteal stromal cells give rise to aggressive osteosarcoma-like lesions upon loss of p53 tumor suppressor through unregulated self-renewal and aberrant osteogenic fates. Therefore, Fgfr3+ endosteal SSCs are abundant in young bone marrow and provide a robust source of osteoblasts, contributing to both normal and aberrant osteogenesis.

Diverse stem cells for periodontal tissue formation and regeneration.

The periodontium is comprised of multiple units of mineralized and nonmineralized tissues including the cementum on the root surface, the alveolar bone, periodontal ligament (PDL), and the gingiva. PDL contains a variety of cell populations including mesenchymal stem/progenitor cells (MSCs) termed PDLSCs, which contribute to periodontal regeneration. Recent studies utilizing mouse genetic models shed light on the identities of these mesenchymal progenitors in their native environment, particularly regarding how they contribute to homeostasis and repair of the periodontium. The current concept is that mesenchymal progenitors in the PDL are localized to the perivascular niche. Single-cell RNA sequencing (scRNA-seq) analyses reveal heterogeneity and cell-type specific markers of cells in the periodontium, as well as their developmental relationship with precursor cells in the dental follicle. The characteristics of PDLSCs and their diversity in vivo are now beginning to be unraveled thanks to insights from mouse genetic models and scRNA-seq analyses, which aid to uncover the fundamental properties of stem cells in the human PDL. The new knowledge will be highly important for developing more effective stem cell-based regenerative therapies to repair periodontal tissues in the future.

Chondrocytes in the resting zone of the growth plate are maintained in a Wnt-inhibitory environment.

Chondrocytes in the resting zone of the postnatal growth plate are characterized by slow cell cycle progression, and encompass a population of parathyroid hormone-related protein (PTHrP)-expressing skeletal stem cells that contribute to the formation of columnar chondrocytes. However, how these chondrocytes are maintained in the resting zone remains undefined. We undertook a genetic pulse-chase approach to isolate slow cycling, label-retaining chondrocytes (LRCs) using a chondrocyte-specific doxycycline-controllable Tet-Off system regulating expression of histone 2B-linked GFP. Comparative RNA-seq analysis identified significant enrichment of inhibitors and activators for Wnt signaling in LRCs and non-LRCs, respectively. Activation of Wnt/ß-catenin signaling in PTHrP+ resting chondrocytes using Pthlh-creER and Apc-floxed allele impaired their ability to form columnar chondrocytes. Therefore, slow-cycling chondrocytes are maintained in a Wnt-inhibitory environment within the resting zone, unraveling a novel mechanism regulating maintenance and differentiation of PTHrP+ skeletal stem cells of the postnatal growth plate.

Angiogenic Effects of Secreted Factors from Periodontal Ligament Stem Cells.

Periodontal disease is a chronic inflammation of tooth-supporting tissues, and the destruction of these tissues results in tooth loss. Regeneration of periodontal tissues is the ultimate goal of periodontal treatment. We previously reported that transplantation of conditioned medium (CM) of periodontal ligament stem cells (PDLSCs) demonstrated the enhancement of periodontal tissue regeneration, compared to CM from fibroblasts (Fibroblast-CM). We hypothesized that the angiogenic effects of PDLSC-CM might participate in the enhanced wound healing of periodontal tissues. The aim of this study was to investigate the effect of PDLSC-CM on the functions of endothelial cells. PDLSCs were cultured from periodontal ligament tissues obtained from healthy volunteers. Human gingival epithelial cells, dermal fibroblasts, osteoblasts, and umbilical vein endothelial cells (HUVECs) were purchased from commercial sources. The functions of endothelial cells were examined using immunostaining of Ki67, observation of nuclear fragmentation and condensation (apoptosis), and network formation on Matrigel. Vascular endothelial cell growth factor (VEGF) level was measured using an ELISA kit. HUVECs demonstrated higher cell viability in PDLSC-CM when compared with those in Fibroblast-CM. HUVECs demonstrated a higher number of Ki67-positive cells and lower apoptosis cells in PDLSC-CM, compared to Fibroblast-CM. Additionally, HUVECs formed more capillary-like structures in PDLSC-CM than Fibroblast-CM. PDLSC-CM contained higher levels of angiogenic growth factor, VEGF, than Fibroblast-CM. Our results showed that PDLSC-CM increased cell viability, proliferation, and capillary formation of HUVECs compared to Fibroblast-CM, suggesting the angiogenic effects of PDLSC-CM, and the effect is a potential regenerative mechanism of periodontal tissues by PDLSC-CM.

A Wnt-mediated transformation of the bone marrow stromal cell identity orchestrates skeletal regeneration.

Bone marrow stromal cells (BMSCs) are versatile mesenchymal cell populations underpinning the major functions of the skeleton, a majority of which adjoin sinusoidal blood vessels and express C-X-C motif chemokine ligand 12 (CXCL12). However, how these cells are activated during regeneration and facilitate osteogenesis remains largely unknown. Cell-lineage analysis using Cxcl12-creER mice reveals that quiescent Cxcl12-creER+ perisinusoidal BMSCs differentiate into cortical bone osteoblasts solely during regeneration. A combined single cell RNA-seq analysis demonstrate that these cells convert their identity into a skeletal stem cell-like state in response to injury, associated with upregulation of osteoblast-signature genes and activation of canonical Wnt signaling components along the single-cell trajectory. ß-catenin deficiency in these cells indeed causes insufficiency in cortical bone regeneration. Therefore, quiescent Cxcl12-creER+ BMSCs transform into osteoblast precursor cells in a manner mediated by canonical Wnt signaling, highlighting a unique mechanism by which dormant stromal cells are enlisted for skeletal regeneration.

Spontaneous differentiation of periodontal ligament stem cells into myofibroblast during ex vivo expansion.

Periodontitis is characterized by the chronic inflammation and destruction of tooth-supporting tissues. Periodontal ligament stem cell (PDLSC) is the mesenchymal stem cell (MSC) population isolated from periodontal ligament, which is the key tissue for regeneration of periodontal tissues. Although transplantation of PDLSCs is proposed as novel regenerative therapy, limited information is available, regarding the characteristic change of PDLSCs during ex vivo expansion. In this study, we encountered morphological change of PDLSCs during standard cell culture and aimed to investigate the change of PDLSCs in stem cell characteristics and to search for the culture condition to maintain stem cell properties. Characteristics of PDLSCs were examined using in vitro osteoblast and adipocyte differentiation. Myofibroblast differentiation was confirmed using immunohistochemistry and collagen gel contraction assay. Replicative senescence was examined by ß-gal staining. PDLSCs changed their morphology from spindle to flat and wide during ex vivo expansion. After the morphological change, PDLSCs showed several features of myofibroblast including extensive stress fiber formation, contraction activity, and myofibroblast marker expression. Upon the morphological change, osteoblastic and adipocyte differentiation capacity were reduced and expression of stem cell-related genes were decreased. ß-Gal staining was not always correlated with the morphological change of PDLSCs. Moreover, exogenous addition of bFGF and PDGF-BB served to maintain spindle shape and osteoblastic differentiation potential of PDLSCs. This study demonstrates that spontaneous differentiation of PDLSCs during ex vivo expansion and may provide the important information of cell culture condition of PDLSCs for clinical use.

Growth Plate Borderline Chondrocytes Behave as Transient Mesenchymal Precursor Cells.

The growth plate provides a substantial source of mesenchymal cells in the endosteal marrow space during endochondral ossification. The current model postulates that a group of chondrocytes in the hypertrophic zone can escape from apoptosis and transform into cells that eventually become osteoblasts in an area beneath the growth plate. The growth plate is composed of cells with various morphologies; particularly at the periphery of the growth plate immediately adjacent to the perichondrium are "borderline" chondrocytes, which align perpendicularly to other chondrocytes. However, in vivo cell fates of these special chondrocytes have not been revealed. Here we show that borderline chondrocytes in growth plates behave as transient mesenchymal precursor cells for osteoblasts and marrow stromal cells. A single-cell RNA-seq analysis revealed subpopulations of Col2a1-creER-marked neonatal chondrocytes and their cell type-specific markers. A tamoxifen pulse to Pthrp-creER mice in the neonatal stage (before the resting zone was formed) preferentially marked borderline chondrocytes. Following the chase, these cells marched into the nascent marrow space, expanded in the metaphyseal marrow, and became Col(2.3 kb)-GFP+ osteoblasts and Cxcl12-GFPhigh reticular stromal "CAR" cells. Interestingly, these borderline chondrocyte-derived marrow cells were short-lived, as they were significantly reduced during adulthood. These findings demonstrate based on in vivo lineage-tracing experiments that borderline chondrocytes in the peripheral growth plate are a particularly important route for producing osteoblasts and marrow stromal cells in growing murine endochondral bones. A special microenvironment neighboring the osteogenic perichondrium might endow these chondrocytes with an enhanced potential to differentiate into marrow mesenchymal cells. © 2019 American Society for Bone and Mineral Research.

Changes in characteristics of periodontal ligament stem cells in spheroid culture.

OBJECTIVES: The periodontal ligament (PDL) has important roles in maintaining homeostasis, wound healing, and regeneration of periodontal tissues by supplying stem/progenitor cells. Periodontal ligament stem cells (PDLSCs) have mesenchymal stem cell (MSC)-like characteristics and can be isolated from periodontal tissues. The aim of this study was to examine the effect of three-dimensional spheroid culture on the characteristics of PDLSCs. MATERIAL AND METHODS: Periodontal ligament stem cells were isolated and cultured from healthy teeth, and PDLSC spheroids were formed by pellet culture in polypropylene tubes. The proliferation of PDLSCs in spheroids and conventional two-dimensional (2D) cultures were examined by immunostaining for Ki67. Cell death and cell size were analyzed using flow cytometry. Gene expression changes were investigated by quantitative real time PCR. RESULTS: Periodontal ligament stem cells spontaneously formed spheroid masses in pellet culture. The size of PDLSC spheroids was inversely proportional to the culture period. Fewer Ki67-positive cells were detected in PDLSC spheroids compared to those in 2D culture. Flow cytometry revealed an increase in dead cells and a decrease in cell size in PDLSC spheroids. The expression levels of genes related to anti-inflammation (TSG6, COX2, MnSOD) and angiogenesis (VEGF, bFGF, HGF) were drastically increased by spheroid culture compared to 2D culture. TSG6 gene expression was inhibited in PDLSC spheroids in the presence of the apoptosis signal inhibitor, Z-VAD-FMK. Additionally, PDLSC spheroid transplantation into rat periodontal defects did not induce the regeneration of periodontal tissues. CONCLUSIONS: We found that spheroid culture of PDLSCs affected several characteristics of PDLSCs, including the expression of genes related to anti-inflammation and angiogenesis; apoptosis signaling may be involved in these changes. Our results revealed the characteristics of PDLSCs in spheroid culture and have provided new information to the field of stem cell research.

The Fate of Transplanted Periodontal Ligament Stem Cells in Surgically Created Periodontal Defects in Rats.

Periodontal disease is chronic inflammation that leads to the destruction of tooth-supporting periodontal tissues. We devised a novel method ("cell transfer technology") to transfer cells onto a scaffold surface and reported the potential of the technique for regenerative medicine. The aim of this study is to examine the efficacy of this technique in periodontal regeneration and the fate of transplanted cells. Human periodontal ligament stem cells (PDLSCs) were transferred to decellularized amniotic membrane and transplanted into periodontal defects in rats. Regeneration of tissues was examined by microcomputed tomography and histological observation. The fate of transplanted PDLSCs was traced using PKH26 and human Alu sequence detection by PCR. Imaging showed more bone in PDLSC-transplanted defects than those in control (amnion only). Histological examination confirmed the enhanced periodontal tissue formation in PDLSC defects. New formation of cementum, periodontal ligament, and bone were prominently observed in PDLSC defects. PKH26-labeled PDLSCs were found at limited areas in regenerated periodontal tissues. Human Alu sequence detection revealed that the level of Alu sequence was not increased, but rather decreased. This study describes a novel stem cell transplantation strategy for periodontal disease using the cell transfer technology and offers new insight for cell-based periodontal regeneration.

Autocrine regulation of mesenchymal progenitor cell fates orchestrates tooth eruption.

Formation of functional skeletal tissues requires highly organized steps of mesenchymal progenitor cell differentiation. The dental follicle (DF) surrounding the developing tooth harbors mesenchymal progenitor cells for various differentiated cells constituting the tooth root-bone interface and coordinates tooth eruption in a manner dependent on signaling by parathyroid hormone-related peptide (PTHrP) and the PTH/PTHrP receptor (PPR). However, the identity of mesenchymal progenitor cells in the DF and how they are regulated by PTHrP-PPR signaling remain unknown. Here, we show that the PTHrP-PPR autocrine signal maintains physiological cell fates of DF mesenchymal progenitor cells to establish the functional periodontal attachment apparatus and orchestrates tooth eruption. A single-cell RNA-seq analysis revealed cellular heterogeneity of PTHrP+ cells, wherein PTHrP+ DF subpopulations abundantly express PPR. Cell lineage analysis using tamoxifen-inducible PTHrP-creER mice revealed that PTHrP+ DF cells differentiate into cementoblasts on the acellular cementum, periodontal ligament cells, and alveolar cryptal bone osteoblasts during tooth root formation. PPR deficiency induced a cell fate shift of PTHrP+ DF mesenchymal progenitor cells to nonphysiological cementoblast-like cells precociously forming the cellular cementum on the root surface associated with up-regulation of Mef2c and matrix proteins, resulting in loss of the proper periodontal attachment apparatus and primary failure of tooth eruption, closely resembling human genetic conditions caused by PPR mutations. These findings reveal a unique mechanism whereby proper cell fates of mesenchymal progenitor cells are tightly maintained by an autocrine system mediated by PTHrP-PPR signaling to achieve functional formation of skeletal tissues.

Cell transfer technology for tissue engineering.

We recently developed novel cell transplantation method "cell transfer technology" utilizing photolithography. Using this method, we can transfer ex vivo expanded cells onto scaffold material in desired patterns, like printing of pictures and letters on a paper. We have investigated the possibility of this novel method for cell-based therapy using several disease models. We first transferred endothelial cells in capillary-like patterns on amnion. The transplantation of the endothelial cell-transferred amnion enhanced the reperfusion in mouse ischemic limb model. The fusion of transplanted capillary with host vessel networks was also observed. The osteoblast- and periodontal ligament stem cell-transferred amnion were next transplanted in bone and periodontal defects models. After healing period, both transplantations improved the regeneration of bone and periodontal tissues, respectively. This method was further applicable to transfer of multiple cell types and the transplantation of osteoblasts and periodontal ligament stem cell-transferred amnion resulted in the improved bone regeneration compared with single cell type transplantation. These data suggested the therapeutic potential of the technology in cell-based therapies for reperfusion of ischemic limb and regeneration of bone and periodontal tissues. Cell transfer technology is applicable to wide range of regenerative medicine in the future.

Conditioned Medium from Periodontal Ligament Stem Cells Enhances Periodontal Regeneration.

Periodontal disease is one of the most common infectious diseases in adults and is characterized by the destruction of tooth-supporting tissues. Mesenchymal stem cells (MSCs) comprise the mesoderm-originating stem cell population, which has been studied and used for cell therapy. However, because of the lower rate of cell survival after MSC transplantation in various disease models, paracrine functions of MSCs have been receiving increased attention as a regenerative mechanism. The aim of this study was to investigate the regenerative potential of transplanted conditioned medium (CM) obtained from cultured periodontal ligament stem cells (PDLSCs), the adult stem cell population in tooth-supporting tissues, using a rat periodontal defect model. Cell-free CM was collected from PDLSCs and fibroblasts, using ultrafiltration and transplanted into surgically created periodontal defects. Protein content of CM was examined by antibody arrays. Formation of new periodontal tissues was analyzed using microcomputed tomography and histological sections. PDLSC-CM transplantation enhanced periodontal tissue regeneration in a concentration-dependent manner, whereas fibroblast-CM did not show any regenerative function. Proteomic analysis revealed that extracellular matrix proteins, enzymes, angiogenic factors, growth factors and cytokines were contained in PDLSC-CM. Furthermore, PDLSC-CM transplantation resulted in the decreased mRNA level of tumor necrosis factor-α (TNF-α) in healing periodontal tissues. In addition, we found that PDLSC-CM suppressed the mRNA level of TNF-α in the monocyte/macrophage cell line, RAW cells, stimulated with IFN-γ. Our findings suggested that PDLSC-CM enhanced periodontal regeneration by suppressing the inflammatory response through TNF-α production, and transplantation of PDLSC-CM could be a novel approach for periodontal regenerative therapy.

Double-layered cell transfer technology for bone regeneration.

For cell-based medicine, to mimic in vivo cellular localization, various tissue engineering approaches have been studied to obtain a desirable arrangement of cells on scaffold materials. We have developed a novel method of cell manipulation called "cell transfer technology", enabling the transfer of cultured cells onto scaffold materials, and controlling cell topology. Here we show that using this technique, two different cell types can be transferred onto a scaffold surface as stable double layers or in patterned arrangements. Various combinations of adherent cells were transferred to a scaffold, amniotic membrane, in overlapping bilayers (double-layered cell transfer), and transferred cells showed stability upon deformations of the material including folding and trimming. Transplantation of mesenchymal stem cells from periodontal ligaments (PDLSC) and osteoblasts, using double-layered cell transfer significantly enhanced bone formation, when compared to single cell type transplantation. Our findings suggest that this double-layer cell transfer is useful to produce a cell transplantation material that can bear two cell layers. Moreover, the transplantation of an amniotic membrane with PDLSCs/osteoblasts by cell transfer technology has therapeutic potential for bone defects. We conclude that cell transfer technology provides a novel and unique cell transplantation method for bone regeneration.