Chenshuang Li

Brief Report: Human Perivascular Stem Cells and Nel‐Like Protein‐1 Synergistically Enhance Spinal Fusion in Osteoporotic Rats

Autologous bone grafts (ABGs) are considered as the gold standard for spinal fusion. However, osteoporotic patients are poor candidates for ABGs due to limited osteogenic stem cell numbers and function of the bone microenvironment. There is a need for stem cell‐based spinal fusion of proven efficacy under either osteoporotic or nonosteoporotic conditions. The purpose of this study is to determine the efficacy of human perivascular stem cells (hPSCs), a population of mesenchymal stem cells isolated from adipose tissue, in the presence and absence of NELL‐1, an osteogenic protein, for spinal fusion in the osteoporosis. Osteogenic differentiation of hPSCs with and without NELL‐1 was tested in vitro. The results indicated that NELL‐1 significantly increased the osteogenic potential of hPSCs in both osteoporotic and nonosteoporotic donors. Next, spinal fusion was performed by implanting scaffolds with regular or high doses of hPSCs, with or without NELL‐1 in ovariectomized rats (n = 41). Regular doses of hPSCs or NELL‐1 achieved the fusion rates of only 20%–37.5% by manual palpation. These regular doses had previously been shown to be effective in nonosteoporotic rat spinal fusion. Remarkably, the high dose of hPSCs+NELL‐1 significantly improved the fusion rates among osteoporotic rats up to approximately 83.3%. Microcomputed tomography imaging and quantification further confirmed solid bony fusion with high dose hPSCs+NELL‐1. Finally, histologically, direct in situ involvement of hPSCs in ossification was shown using undecalcified samples. To conclude, hPSCs combined with NELL‐1 synergistically enhances spinal fusion in osteoporotic rats and has great potential as a novel therapeutic strategy for osteoporotic patients. Stem Cells 2015;33:3158–3163

Proliferation and Osteogenic Differentiation of Mesenchymal Stem Cells Induced by a Short Isoform of NELL‐1

Neural epidermal growth factor‐like (NEL)‐like protein 1 (NELL‐1) has been identified as an osteoinductive differentiation factor that promotes mesenchymal stem cell (MSC) osteogenic differentiation. In addition to full‐length NELL‐1, there are several NELL‐1‐related transcripts reported. We used rapid amplification of cDNA ends to recover potential cDNA of NELL‐1 isoforms. A NELL‐1 isoform with the N‐terminal 240 amino acid (aa) residues truncated was identified. While full‐length NELL‐1 that contains 810 aa residues (NELL‐1810) plays an important role in embryologic skeletal development, the N‐terminal‐truncated NELL‐1 isoform (NELL‐1570) was expressed postnatally. Similar to NELL‐1810, NELL‐1570 induced MSC osteogenic differentiation. In addition, NELL‐1570 significantly stimulated MSC proliferation in multiple MSC‐like populations such as murine C3H10T1/2 MSC cell line, mouse primary MSCs, and perivascular stem cells, which is a type of stem cells proposed as the perivascular origin of MSCs. In contrast, NELL‐1810 demonstrated only limited stimulation of MSC proliferation. Similar to NELL‐1810, NELL‐1570 was found to be secreted from host cells. Both NELL‐1570 expression lentiviral vector and column‐purified recombinant protein NELL‐1570 demonstrated almost identical effects in MSC proliferation and osteogenic differentiation, suggesting that NELL‐1570 may function as a pro‐osteogenic growth factor. In vivo, NELL‐1570 induced significant calvarial defect regeneration accompanied by increased cell proliferation. Thus, NELL‐1570 has the potential to be used for cell‐based or hormone‐based therapy of bone regeneration. Stem Cells 2015;33:904–915

Fibromodulin reduces scar formation in adult cutaneous wounds by eliciting a fetal-like phenotype

Blocking transforming growth factor (TGF)β1 signal transduction has been a central strategy for scar reduction; however, this approach appears to be minimally effective. Here, we show that fibromodulin (FMOD), a 59-kD small leucine-rich proteoglycan critical for normal collagen fibrillogenesis, significantly reduces scar formation while simultaneously increasing scar strength in both adult rodent models and porcine wounds, which simulate human cutaneous scar repair. Mechanistically, FMOD uncouples pro-migration/contraction cellular signals from pro-fibrotic signaling by selectively enhancing SMAD3-mediated signal transduction, while reducing AP-1-mediated TGFβ1 auto-induction and fibrotic extracellular matrix accumulation. Consequently, FMOD accelerates TGFβ1-responsive adult fibroblast migration, myofibroblast conversion, and function. Furthermore, our findings strongly indicate that, by delicately orchestrating TGFβ1 activities rather than indiscriminately blocking TGFβ1, FMOD elicits fetal-like cellular and molecular phenotypes in adult dermal fibroblasts in vitro and adult cutaneous wounds in vivo, which is a unique response of living system undescribed previously. Taken together, this study illuminates the signal modulating activities of FMOD beyond its structural support functions, and highlights the potential for FMOD-based therapies to be used in cutaneous wound repair.

Fibromodulin reduces scar size and increases scar tensile strength in normal and excessive-mechanical-loading porcine cutaneous wounds

Hypertrophic scarring is a major postoperative complication which leads to severe disfigurement and dysfunction in patients and usually requires multiple surgical revisions due to its high recurrence rates. Excessive‐mechanical‐loading across wounds is an important initiator of hypertrophic scarring formation. In this study, we demonstrate that intradermal administration of a single extracellular matrix (ECM) molecule—fibromodulin (FMOD) protein—can significantly reduce scar size, increase tensile strength, and improve dermal collagen architecture organization in the normal and even excessive‐mechanical‐loading red Duroc pig wound models. Since pig skin is recognized by the Food and Drug Administration as the closest animal equivalent to human skin, and because red Duroc pigs show scarring that closely resembles human proliferative scarring and hypertrophic scarring, FMOD‐based technologies hold high translational potential and applicability to human patients suffering from scarring—especially hypertrophic scarring.

hPSCs and NELL-1 Synergistically Enhance Spinal Fusion in Osteoporotic Rats

Autologous bone grafts (ABGs) are considered as the gold standard for spinal fusion. However, osteoporotic patients are poor candidates for ABGs due to limited osteogenic stem cell numbers and function of the bone microenvironment. There is a need for stem cell‐based spinal fusion of proven efficacy under either osteoporotic or nonosteoporotic conditions. The purpose of this study is to determine the efficacy of human perivascular stem cells (hPSCs), a population of mesenchymal stem cells isolated from adipose tissue, in the presence and absence of NELL‐1, an osteogenic protein, for spinal fusion in the osteoporosis. Osteogenic differentiation of hPSCs with and without NELL‐1 was tested in vitro. The results indicated that NELL‐1 significantly increased the osteogenic potential of hPSCs in both osteoporotic and nonosteoporotic donors. Next, spinal fusion was performed by implanting scaffolds with regular or high doses of hPSCs, with or without NELL‐1 in ovariectomized rats (n = 41). Regular doses of hPSCs or NELL‐1 achieved the fusion rates of only 20%–37.5% by manual palpation. These regular doses had previously been shown to be effective in nonosteoporotic rat spinal fusion. Remarkably, the high dose of hPSCs+NELL‐1 significantly improved the fusion rates among osteoporotic rats up to approximately 83.3%. Microcomputed tomography imaging and quantification further confirmed solid bony fusion with high dose hPSCs+NELL‐1. Finally, histologically, direct in situ involvement of hPSCs in ossification was shown using undecalcified samples. To conclude, hPSCs combined with NELL‐1 synergistically enhances spinal fusion in osteoporotic rats and has great potential as a novel therapeutic strategy for osteoporotic patients. Stem Cells 2015;33:3158–3163

Neurexin Superfamily Cell Membrane Receptor Contactin-Associated Protein Like-4 (Cntnap4) Is Involved in Neural EGFL-Like 1 (Nell-1)-Responsive Osteogenesis: CNTNAP4 INVOLVED IN NELL-1-RESPONSIVE OSTEOGENESIS

Contactin‐associated protein‐like 4 (Cntnap4) is a member of the neurexin superfamily of transmembrane molecules that have critical functions in neuronal cell communication. Cntnap4 knockout mice display decreased presynaptic gamma‐aminobutyric acid (GABA) and increased dopamine release that is associated with severe, highly penetrant, repetitive, and perseverative movements commonly found in human autism spectrum disorder patients. However, no known function of Cntnap4 has been revealed besides the nervous system. Meanwhile, secretory protein neural EGFL‐like 1 (Nell‐1) is known to exert potent osteogenic effects in multiple small and large animal models without the off‐target effects commonly found with bone morphogenetic protein 2. In this study, while searching for a Nell‐1‐specific cell surface receptor during osteogenesis, we identified and validated a ligand/receptor‐like interaction between Nell‐1 and Cntnap4 by demonstrating: 1) Nell‐1 and Cntnap4 colocalization on the surface of osteogenic‐committed cells; 2) high‐affinity interaction between Nell‐1 and Cntnap4; 3) abrogation of Nell‐1‐responsive Wnt and MAPK signaling transduction, as well as osteogenic effects, via Cntnap4 knockdown; and 4) replication of calvarial cleidocranial dysplasias‐like defects observed in Nell‐1‐deficient mice in Wnt1‐Cre‐mediated Cntnap4‐knockout transgenic mice. In aggregate, these findings indicate that Cntnap4 plays a critical role in Nell‐1‐responsive osteogenesis. Further, this is the first functional annotation for Cntnap4 in the musculoskeletal system. Intriguingly, Nell‐1 and Cntnap4 also colocalize on the surface of human hippocampal interneurons, implicating Nell‐1 as a potential novel ligand for Cntnap4 in the nervous system. This unexpected characterization of the ligand/receptor‐like interaction between Nell‐1 and Cntnap4 indicates a novel biological functional axis for Nell‐1 and Cntnap4 in osteogenesis and, potentially, in neural development and function.

Nfatc1 Is a Functional Transcriptional Factor Mediating Nell-1-Induced Runx3 Upregulation in Chondrocytes

Neural EGFL like 1 (Nell-1) is essential for chondrogenic differentiation, maturation, and regeneration. Our previous studies have demonstrated that Nell-1’s pro-chondrogenic activities are predominantly reliant upon runt-related transcription factor 3 (Runx3)-mediated Indian hedgehog (Ihh) signaling. Here, we identify the nuclear factor of activated T-cells 1 (Nfatc1) as the key transcriptional factor mediating the Nell-1 → Runx3 signal transduction in chondrocytes. Using chromatin immunoprecipitation assay, we were able to determine that Nfatc1 binds to the −833–−810 region of the Runx3-promoter in response to Nell-1 treatment. By revealing the Nell-1 → Nfatc1 → Runx3 → Ihh cascade, we demonstrate the involvement of Nfatc1, a nuclear factor of activated T-cells, in chondrogenesis, while providing innovative insights into developing a novel therapeutic strategy for cartilage regeneration and other chondrogenesis-related conditions.

Brief Report: Human Perivascular Stem Cells andNel-Like Protein-1 Synergistically Enhance Spinal Fusion in Osteoporotic Rats: hPSCs and NELL-1 in Spinal Fusion for Osteoporosis

Autologous bone grafts (ABGs) are considered as the gold standard for spinal fusion. However, osteoporotic patients are poor candidates for ABGs due to limited osteogenic stem cell numbers and function of the bone microenvironment. There is a need for stem cell‐based spinal fusion of proven efficacy under either osteoporotic or nonosteoporotic conditions. The purpose of this study is to determine the efficacy of human perivascular stem cells (hPSCs), a population of mesenchymal stem cells isolated from adipose tissue, in the presence and absence of NELL‐1, an osteogenic protein, for spinal fusion in the osteoporosis. Osteogenic differentiation of hPSCs with and without NELL‐1 was tested in vitro. The results indicated that NELL‐1 significantly increased the osteogenic potential of hPSCs in both osteoporotic and nonosteoporotic donors. Next, spinal fusion was performed by implanting scaffolds with regular or high doses of hPSCs, with or without NELL‐1 in ovariectomized rats (n = 41). Regular doses of hPSCs or NELL‐1 achieved the fusion rates of only 20%–37.5% by manual palpation. These regular doses had previously been shown to be effective in nonosteoporotic rat spinal fusion. Remarkably, the high dose of hPSCs+NELL‐1 significantly improved the fusion rates among osteoporotic rats up to approximately 83.3%. Microcomputed tomography imaging and quantification further confirmed solid bony fusion with high dose hPSCs+NELL‐1. Finally, histologically, direct in situ involvement of hPSCs in ossification was shown using undecalcified samples. To conclude, hPSCs combined with NELL‐1 synergistically enhances spinal fusion in osteoporotic rats and has great potential as a novel therapeutic strategy for osteoporotic patients. Stem Cells 2015;33:3158–3163