Curculigoside Ameliorates Bone Loss by Influencing Mesenchymal Stem Cell Fate in Aging Mice

Senile osteoporosis is characterized by increased bone loss and fat accumulation in marrow. Curculigoside (CCG) is the major bioactive component of Curculigo orchioides, which has been used as anti-osteoporosis therapy for elder patients since antiquity. We aimed to investigate the underlying mechanisms by which CCG regulated the bone-fat balance in marrow of aging mice. In our study, CCG treatment was identified to interfere with the stem cell lineage commitment both in vivo and in vitro. In vivo, CCG promoted the transcriptional co-activator with PDZ-binding motif (TAZ) expression to reverse age-related bone loss and marrow adiposity. In vitro, proper concentration of CCG upregulated TAZ expression to increase osteogenesis and decrease adipogenesis of bone marrow mesenchymal stem cells (BMSCs). This regulating effect was discounted by TAZ knockdown or the use of MEK-ERK pathway inhibitor, UO126. Above all, our study confirmed the rescuing effects of CCG on the differential shift from adipogenesis to osteogenesis of BMSCs in aging mice and provided a scientific basis for the clinical use of CCG in senile osteoporosis.

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Effect of the 3D Artificial Nichoid on the Morphology and Mechanobiological Response of Mesenchymal Stem Cells Cultured In Vitro

Cells ◽

10.3390/cells9081873 ◽

2020 ◽

Vol 9 (8) ◽

pp. 1873 ◽

Cited By ~ 3

Author(s):

Andrea Remuzzi ◽

Barbara Bonandrini ◽

Matteo Tironi ◽

Lorena Longaretti ◽

Marina Figliuzzi ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Cultured Cells ◽

Three Dimensions ◽

Nuclear Pore ◽

Cells Cultured

Stem cell fate and behavior are affected by the bidirectional communication of cells and their local microenvironment (the stem cell niche), which includes biochemical cues, as well as physical and mechanical factors. Stem cells are normally cultured in conventional two-dimensional monolayer, with a mechanical environment very different from the physiological one. Here, we compare culture of rat mesenchymal stem cells on flat culture supports and in the “Nichoid”, an innovative three-dimensional substrate micro-engineered to recapitulate the architecture of the physiological niche in vitro. Two versions of the culture substrates Nichoid (single-layered or “2D Nichoid” and multi-layered or “3D Nichoid”) were fabricated via two-photon laser polymerization in a biocompatible hybrid organic-inorganic photoresist (SZ2080). Mesenchymal stem cells, isolated from rat bone marrow, were seeded on flat substrates and on 2D and 3D Nichoid substrates and maintained in culture up to 2 weeks. During cell culture, we evaluated cell morphology, proliferation, cell motility and the expression of a panel of 89 mesenchymal stem cells’ specific genes, as well as intracellular structures organization. Our results show that mesenchymal stem cells adhered and grew in the 3D Nichoid with a comparable proliferation rate as compared to flat substrates. After seeding on flat substrates, cells displayed large and spread nucleus and cytoplasm, while cells cultured in the 3D Nichoid were spatially organized in three dimensions, with smaller and spherical nuclei. Gene expression analysis revealed the upregulation of genes related to stemness and to mesenchymal stem cells’ features in Nichoid-cultured cells, as compared to flat substrates. The observed changes in cytoskeletal organization of cells cultured on 3D Nichoids were also responsible for a different localization of the mechanotransducer transcription factor YAP, with an increase of the cytoplasmic retention in cells cultured in the 3D Nichoid. This difference could be explained by alterations in the import of transcription factors inside the nucleus due to the observed decrease of mean nuclear pore diameter, by transmission electron microscopy. Our data show that 3D distribution of cell volume has a profound effect on mesenchymal stem cells structure and on their mechanobiological response, and highlight the potential use of the 3D Nichoid substrate to strengthen the potential effects of MSC in vitro and in vivo.

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Deterministic Trigger of Self-Renewal in Expanded HSCs Expressing Hoxb4 + Antisense Pbx1.

Blood ◽

10.1182/blood.v106.11.800.800 ◽

2005 ◽

Vol 106 (11) ◽

pp. 800-800

Author(s):

Sonia Cellot ◽

Jana Krosl ◽

Keith Humphries ◽

Guy Sauvageau

Keyword(s):

Cell Proliferation ◽

Stem Cell ◽

Cell Fate ◽

Time Course ◽

Cell Cycle Distribution ◽

Self Renewal ◽

Primary Mouse ◽

The Impact

Abstract We previously reported the generation of pluripotent and ultracompetitive HSCs through modulation of Hoxb4 and Pbx1 levels. These Hoxb4hiPbx1lo HSCs display a tremendous regenerative potential, yet they are still fully responsive to in vivo regulatory signals that control stem cell pool size (20 000 HSCmouse) and differentiation pathways. Further work in our laboratory attempted to circumvent these physiological constraints by expanding Hoxb4hiPbx1lo transduced HSCs in vitro, and hence revealing their intrinsic expansion potential. Independent experiments were performed where primary mouse BM cells were co-infected with retroviruses encoding antisense Pbx1 cDNA plus YFP, and Hoxb4 plus GFP (double gene transfer ranged between 20–50%). Hoxb4hiPbx1lo HSCs measured using the CRU assay expanded by 105-fold during a 12 day in vitro culture. Following serial transplantations, these cells displayed an additional 4–5 log expansion in vivo. Total stem cell content per animal remained within normal limits. Southern blot analyses of proviral integrations showed that the expansion was polyclonal, and analyses of individually expanded clones provided a molecular proof of in vitro self-renewal (SR). This unprecedented level of HSC expansion in such a short time course (105-fold in 12 days) implies an absolute HSC doubling time of approximately 17 hours in our culture, raising the possibility that virtually all dividing HSCs undergo self-renewal. This analysis prompted us to dissect the impact of Hoxb4 on cell proliferation versus cell fate (SR?). When analyzed during the period of maximal HSC expansion, the cell cycle distribution of Sca+ or Sca+Lin− cells were comparable between the cultures initiated with neo control versus Hoxb4 BM cells (CTL vs Hoxb4: G0/G1: 66% vs 83%; S: 15% vs 9%; G2/M: 18% vs 7%). Correspondingly, CFSE tracking studies confirmed the identical, or even lower, number of cellular divisions in Sca+ cells isolated from cultures initiated with Hoxb4 versus neo transduced cells. Annexin V studies precluded protection from apoptosis as the major mechanism to increase HSC numbers since similar results (3–10% positive cells) were observed in the Hoxb4 versus neo-transduced cells. In summary, our studies support the emerging concept that distinct molecular pathways regulate cell proliferation and self-renewal, suggesting that Hoxb4 + antisense Pbx1 predominantly triggers self-renewal over HSC proliferation.

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Constructing stem cell microenvironments using bioengineering approaches

Physiological Genomics ◽

10.1152/physiolgenomics.00099.2013 ◽

2013 ◽

Vol 45 (23) ◽

pp. 1123-1135 ◽

Cited By ~ 29

Author(s):

David A. Brafman

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Cell Fate ◽

Disease Modeling ◽

Mechanical Stimuli ◽

Culture Methods ◽

Stem Cell Fate ◽

And Function

Within the adult organism, stem cells reside in defined anatomical microenvironments called niches. These architecturally diverse microenvironments serve to balance stem cell self-renewal and differentiation. Proper regulation of this balance is instrumental to tissue repair and homeostasis, and any imbalance can potentially lead to diseases such as cancer. Within each of these microenvironments, a myriad of chemical and physical stimuli interact in a complex (synergistic or antagonistic) manner to tightly regulate stem cell fate. The in vitro replication of these in vivo microenvironments will be necessary for the application of stem cells for disease modeling, drug discovery, and regenerative medicine purposes. However, traditional reductionist approaches have only led to the generation of cell culture methods that poorly recapitulate the in vivo microenvironment. To that end, novel engineering and systems biology approaches have allowed for the investigation of the biological and mechanical stimuli that govern stem cell fate. In this review, the application of these technologies for the dissection of stem cell microenvironments will be analyzed. Moreover, the use of these engineering approaches to construct in vitro stem cell microenvironments that precisely control stem cell fate and function will be reviewed. Finally, the emerging trend of using high-throughput, combinatorial methods for the stepwise engineering of stem cell microenvironments will be explored.

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Melanin as an Endogenous MRI Contrast Agent for Stem Cell Delivery in Stroke

10.21203/rs.3.rs-466208/v1 ◽

2021 ◽

Author(s):

Randy Chang ◽

Jesse Jones

Keyword(s):

Stem Cell ◽

Neural Progenitor Cells ◽

Fluorescent Microscopy ◽

Cell Lineage ◽

Mri Contrast Agent ◽

Cmv Promoter ◽

Antibody Staining ◽

Stem Cell Delivery

Abstract In vivo measures of survival, growth, and migration of stem cell transplants for stroke therapy remain inadequate. Since such biomarkers in the central nervous system do not exist, we sought to investigate the potential of melanin as an endogenous MR contrast medium for monitoring cell lineage transplants. Expression of a human gene, tyrosinase, induces melanogenesis, but unlike particle-based tracking agents, tyrosinase incorporated into the host genome will be replicated during mitosis.Tyrosinase and the gene for a co-enzyme, tyrosinase-related protein 1, were placed under the CMV promoter. The construct was inserted into 293 HEK (human embryonal kidney) and iPS NPC (induced pluripotent neural progenitor) cells via plasmid transfection and viral transduction, respectively. Stroked mice were injected with melanin-producing cells. Control mice were injected with native cell lines. In vitro expression was measured by fluorescent microscopy, immunocytochemistry, spectroscopy, PCR, and MRI.Robust in vitro melanin production was achieved in both cell lineages demonstrated by significant T1 shortening on in vivo MRI. Pathologic correlation demonstrated colocalization of pigmented regions in the injection sites with human antinuclear antibody staining. Through induction of melanogenesis that is reproducible across multiple cell divisions, MR-based imaging of clinically relevant cell lineage transplants is possible.

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Clinical-grade stem cell–derived retinal pigment epithelium patch rescues retinal degeneration in rodents and pigs

Science Translational Medicine ◽

10.1126/scitranslmed.aat5580 ◽

2019 ◽

Vol 11 (475) ◽

pp. eaat5580 ◽

Cited By ~ 57

Author(s):

Ruchi Sharma ◽

Vladimir Khristov ◽

Aaron Rising ◽

Balendu Shekhar Jha ◽

Roba Dejene ◽

...

Keyword(s):

Stem Cell ◽

Retinal Pigment Epithelium ◽

Pigment Epithelium ◽

Retinal Pigment ◽

Age Related Macular Degeneration ◽

Clinical Grade ◽

Functional Validation ◽

Age Related

Considerable progress has been made in testing stem cell–derived retinal pigment epithelium (RPE) as a potential therapy for age-related macular degeneration (AMD). However, the recent reports of oncogenic mutations in induced pluripotent stem cells (iPSCs) underlie the need for robust manufacturing and functional validation of clinical-grade iPSC-derived RPE before transplantation. Here, we developed oncogenic mutation-free clinical-grade iPSCs from three AMD patients and differentiated them into clinical-grade iPSC-RPE patches on biodegradable scaffolds. Functional validation of clinical-grade iPSC-RPE patches revealed specific features that distinguished transplantable from nontransplantable patches. Compared to RPE cells in suspension, our biodegradable scaffold approach improved integration and functionality of RPE patches in rats and in a porcine laser-induced RPE injury model that mimics AMD-like eye conditions. Our results suggest that the in vitro and in vivo preclinical functional validation of iPSC-RPE patches developed here might ultimately be useful for evaluation and optimization of autologous iPSC-based therapies.

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Regulating Fibrocartilage Stem Cells via TNF-α/Nf-κB in TMJ Osteoarthritis

Journal of Dental Research ◽

10.1177/00220345211037248 ◽

2021 ◽

pp. 002203452110372

Author(s):

R. Bi ◽

K. Chen ◽

Y. Wang ◽

X. Luo ◽

Q. Li ◽

...

Keyword(s):

Stem Cell ◽

Cell Lineage ◽

Cartilage Degeneration ◽

Lineage Tracing ◽

Sprague Dawley ◽

Etanercept Treatment ◽

Tnf Α ◽

Temporomandibular Joint Osteoarthritis

In this study, we investigate harnessing fibrocartilage stem cell (FCSC) capacities by regulating tumor necrosis factor α (TNF-α) signaling for cartilage repair in temporomandibular joint osteoarthritis (TMJOA). Stem cell specifics for FCSCs were characterized in the presence of TNF-α. Etanercept as a TNF-α inhibitor and BAY 11-7082 as an Nf-κB inhibitor were used to study TNF-α regulation of FCSCs. Lineage tracing was performed in Gli1-CreERT+;Tmfl/fl mice when etanercept (1 mg/kg, every 3 d) or isometric vehicle was subcutaneously injected to trace specific changes in FCSCs. Surgically induced TMJOA Sprague-Dawley rats were generated with BAY 11-7082 (5 mg/kg, every 3 d) or vehicle subcutaneous injection to investigate the functional role of TNF-α/Nf-κB in TMJOA. Anterior disc displacement (ADD) rabbits were used to analyze the therapeutic effect of etanercept as a TMJOA intra-articular treatment with etanercept (0.02 mg in 100 μL, every 2 wk) or isometric vehicle. In vitro, TNF-α inhibited proliferation of FCSCs and increased FCSC apoptosis. TNF-α activation interfered with osteogenic and chondrogenic differentiation of FCSCs, while etanercept could partially recover FCSC specificity from TNF-α. FCSC lineage tracing in Gli1-CreERT+;Tmfl/fl mice showed that the chondrogenic capacity of Gli1+ cell lineage was markedly suppressed in osteoarthritis cartilage, the phenotype of which could be significantly rescued by etanercept. Specifically blocking the Nf-κB pathway could significantly weaken the regulatory effect of TNF-α on FCSC specificity in vitro and in TMJOA rats in vivo. Finally, intra-articular etanercept treatment efficiently rescued TMJ cartilage degeneration and growth retardation in ADD rabbits. Inhibition of TNF-α signaling reduced Nf-κB transcripts and recovered FCSC specificities. In vivo, etanercept treatment effectively rescued the osteoarthritis phenotype in TMJOA mice and ADD rabbits. These data suggest a novel therapeutic mechanism whereby TNF-α/Nf-κB inhibition promotes FCSC chondrogenic capacity for cartilage transformation in TMJOA.

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Osteoporosis Recovery byAntrodia camphorataAlcohol Extracts through Bone Regeneration in SAMP8 Mice

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2016/2617868 ◽

2016 ◽

Vol 2016 ◽

pp. 1-9 ◽

Cited By ~ 2

Author(s):

Hen-Yu Liu ◽

Chiung-Fang Huang ◽

Chun-Hao Li ◽

Ching-Yu Tsai ◽

Wei-Hong Chen ◽

...

Keyword(s):

Bone Loss ◽

Bone Matrix ◽

Mineral Density ◽

Antrodia Camphorata ◽

Osteoporosis Therapy ◽

Alizarin Red ◽

Alcohol Extract ◽

Samp8 Mice

Antrodia camphoratahas previously demonstrated the efficacy in treating cancer and anti-inflammation. In this study, we are the first to evaluateAntrodia camphorataalcohol extract (ACAE) for osteoporosis recoveryin vitrowith preosteoblast cells (MC3T3-E1) andin vivowith an osteoporosis mouse model established in our previous studies, ovariectomized senescence accelerated mice (OVX-SAMP8). Our results demonstrated that ACAE treatment was slightly cytotoxic to preosteoblast at 25 μg/mL, by which the osteogenic gene expression (RUNX2, OPN, and OCN) was significantly upregulated with an increased ratio of OPG to RANKL, indicating maintenance of the bone matrix through inhibition of osteoclastic pathway. Additionally, evaluation by Alizarin Red S staining showed increased mineralization in ACAE-treated preosteoblasts. Forin vivostudy, our results indicated that ACAE inhibits bone loss and significantly increases percentage bone volume, trabecular bone number, and bone mineral density in OVX-SAMP8 mice treated with ACAE. Collectively,in vitroandin vivoresults showed that ACAE could promote osteogenesis and prevent bone loss and should be considered an evidence-based complementary and alternative medicine for osteoporosis therapy through the maintenance of bone health.

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Angptl2 is a Marker of Cellular Senescence: The Physiological and Pathophysiological Impact of Angptl2-Related Senescence

International Journal of Molecular Sciences ◽

10.3390/ijms222212232 ◽

2021 ◽

Vol 22 (22) ◽

pp. 12232

Author(s):

Nathalie Thorin-Trescases ◽

Pauline Labbé ◽

Pauline Mury ◽

Mélanie Lambert ◽

Eric Thorin

Keyword(s):

Cell Fate ◽

Cellular Senescence ◽

Cellular Response ◽

Inflammatory Diseases ◽

Age Related ◽

A Cell ◽

Future Work ◽

The Impact

Cellular senescence is a cell fate primarily induced by DNA damage, characterized by irreversible growth arrest in an attempt to stop the damage. Senescence is a cellular response to a stressor and is observed with aging, but also during wound healing and in embryogenic developmental processes. Senescent cells are metabolically active and secrete a multitude of molecules gathered in the senescence-associated secretory phenotype (SASP). The SASP includes inflammatory cytokines, chemokines, growth factors and metalloproteinases, with autocrine and paracrine activities. Among hundreds of molecules, angiopoietin-like 2 (angptl2) is an interesting, although understudied, SASP member identified in various types of senescent cells. Angptl2 is a circulatory protein, and plasma angptl2 levels increase with age and with various chronic inflammatory diseases such as cancer, atherosclerosis, diabetes, heart failure and a multitude of age-related diseases. In this review, we will examine in which context angptl2 was identified as a SASP factor, describe the experimental evidence showing that angptl2 is a marker of senescence in vitro and in vivo, and discuss the impact of angptl2-related senescence in both physiological and pathological conditions. Future work is needed to demonstrate whether the senescence marker angptl2 is a potential clinical biomarker of age-related diseases.

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HDAC6 inactivates Runx2 promoter to block osteogenesis of bone marrow stromal cells in age-related bone loss of mice

Stem Cell Research & Therapy ◽

10.1186/s13287-021-02545-w ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Chao Ma ◽

Juan Gao ◽

Jun Liang ◽

Weixiang Dai ◽

Zhenfei Wang ◽

...

Keyword(s):

Bone Loss ◽

Bone Formation ◽

Osteogenic Differentiation ◽

Differentiation Potential ◽

Runx2 Expression ◽

Aged Mice ◽

Age Related ◽

Old Mice

Abstract Background Senile osteoporosis can cause bone fragility and increased risk for fractures and has been one of the most prevalent and severe diseases affecting the elderly population worldwidely. The underlying mechanisms are currently intensive areas of investigation. In age-related bone loss, decreased bone formation overweighs increased bone resorption. The molecular mechanisms underlying defective bone formation in age-related bone loss are not completely understood. In particular, the specific role of histone acetylation in age-related bone loss has not been examined thoroughly. Methods We employed 6- and 18-month-old mice to investigate the mechanisms of defective bone formation in age-related bone loss. Bone marrow stromal cells (BMSCs) were induced to undergo in vitro osteogenic differentiation. Chromatin immunoprecipitation (ChIP) was used to investigate the binding of histone deacetylases (HDACs) on Runx2 promoter in BMSCs. Luciferase reporter and transient transfection assay were employed to study Runx2 gene expression modulation by HDAC and androgen receptor (AR). siRNA and HDAC6 inhibitor, Tubastatin A, were used to inhibit HDAC6 in vitro. And systemic administration of Tubastatin A was used to block HDAC6 in vivo. Results Age-related trabecular bone loss was observed in 18-month-old mice compared with 6-month-old mice. In vitro osteogenic differentiation potential of BMSCs from 18-month-old mice was weaker than 6-month-old mice, in which there was Runx2 expression inactivation in BMSCs of 18-month-old mice compared with 6-month-old mice, which was attributable to HDAC6-mediated histone hypoacetylation in Runx2 promoter. There was competitive binding of HDAC6 and AR on Runx2 promoter to modulate Runx2 expression in BMSCs. More importantly, through siRNA- or specific inhibitor-mediated HDAC6 inhibition, we could activate Runx2 expression, rescue in vitro osteogenesis potential of BMSCs, and alleviate in vivo age-related bone loss of mice. Conclusion HDAC6 accumulation and histone hypoacetylation on Runx2 promoter contributed to the attenuation of in vitro osteogenic differentiation potential of BMSCs from aged mice. Through HDAC6 inhibition, we could activate Runx2 expression and osteogenic differentiation potential of BMSCs from aged mice and alleviate the age-related bone loss of aged mice. Our study will benefit not only for understanding the age-related bone loss, but also for finding new therapies to treat senile osteoporosis.

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USP7 as part of non-canonical PRC1.1 is a druggable target in leukemia

10.1101/221093 ◽

2017 ◽

Author(s):

Henny Maat ◽

Jennifer Jaques ◽

Aida Rodríguez López ◽

Shanna M. Hogeling ◽

Marcel P. de Vries ◽

...

Keyword(s):

Stem Cell ◽

Cell Fate ◽

Therapeutic Approach ◽

Human Leukemia ◽

List Type ◽

Essential Component ◽

Poor Risk ◽

And Function

ABSTRACTAcute myeloid leukemia (AML) is a highly heterogeneous disease in which genetic and epigenetic changes disturb regulatory mechanisms controlling stem cell fate and maintenance. AML still remains difficult to treat, in particular in poor risk AML patients carrying TP53 mutations. Here, we identify the deubiquitinase USP7 as an integral member of non-canonical PRC1.1 and show that targeting of USP7 provides an alternative therapeutic approach for AML. USP7 inhibitors effectively induced apoptosis in (primary) AML cells, also independent of the USP7-MDM2-TP53 axis, whereby survival of both the cycling as well as quiescent populations was affected. MLL-AF9-induced leukemia was significantly delayed in vivo in human leukemia xenografts. We previously showed that non-canonical PRC1.1 is critically important for leukemic stem cell self-renewal, and that genetic knockdown of the PRC1.1 chromatin binding component KDM2B abrogated leukemia development in vitro and in vivo [1]. Here, by performing KDM2B interactome studies in TP53mut cells we identify that USP7 is an essential component of PRC1.1 and is required for its stability and function. USP7 inhibition results in disassembly of the PRC1.1 complex and consequently loss of binding to its target loci. Loss of PRC1.1 binding coincided with reduced H2AK119ub and H3K27ac levels and diminished gene transcription, whereas H3K4me3 levels remained unaffected. Our studies highlight the diverse functions of USP7 and link it to Polycomb-mediated epigenetic control. USP7 inhibition provides an efficient therapeutic approach for AML, also in the most aggressive subtypes with mutations in TP53.Key pointsUSP7 is a therapeutic target in leukemia, including poor risk TP53mut AML.USP7 is an essential component of non-canonical PRC1.1 and is required for its stability and function.

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