Promijenjeni kanonijski signalni put Hedgehog-Gli kod pesticidima inducirane aplazije koštane srži ispitan na mišjem modelu

The mechanistic interplay between pesticide exposure and development of marrow aplasia is not yet well established but there are indices that chronic pesticide exposure in some instances causes marrow aplasia like haematopoietic degenerative condition in human beings. Canonical Hedgehog (Hh) signalling has multiple roles in a wide range of developmental processes, including haematopoiesis. The present study was designed to explore the status of four important components of the canonical Hedgehog signalling cascade, the Sonic Hedgehog (Shh), Ptch1, Smo, and Gli1, in a mouse model of chronic pesticide-induced bone marrow aplasia. We used 5 % aqueous mixture of pesticides (chlorpyriphos, prophenophos, cypermethrin, alpha-methrin, and hexaconazole) for inhalation and dermal exposure of 6 hours per day and 5 days a week up to 90 days. Murine bone marrow aplasia related to chronic pesticide treatment was confi rmed primarily by haemogram, bone marrow cellularity, short term bone marrow explant culture for cellular kinetics, bone marrow smear, and fl ow cytometric Lin-Sca-1+C-kit+ extracellular receptor expression pattern. Later, components of hed ehog signalling were analysed in the bone marrow of both control and pesticide-treated aplastic groups of animals. The results depicted pancytopenic feature of peripheral blood, developmental anomaly of neutrophils, depression of primitive stem and progenitor population along with Shh, Ptch1, Smo and Gli1 expression in aplasia group. This investigation suggests that pesticide-induced downregulation of two critically important proteins - Ptch1 and Gli1 - inside the haematopoietic stem and progenitor cell population impairs haematopoietic homeostasis and regeneration mechanism in vivo concurrent with bone marrow aplasia.Mehaničko međudjelovanje izlaganja pesticidima i razvoja aplazije koštane srži još uvijek nije u potpunosti utvrđeno, ali postoje naznake da kronično izlaganje pesticidima u nekim slučajevima može uzrokovati aplaziju koštane srži poput hematopoetskoga degenerativnog stanja u ljudi. Kanonijski signalni put Hedgehog (Hh) ima višestruke uloge u mnogim razvojnim procesima, uključujući i hematopoezu. Ovo je ispitivanje imalo za cilj istražiti status četiri glavne sastavnice kanonijskoga signalnoga puta Hedgehog, Sonic Hedgehog (Shh), Ptch1, Smo i Gli1, na mišjem modelu pesticidima inducirane aplazije koštane srži. Koristili smo 5 % vodenu mješavinu pesticida (klorpirifos, profenofos, cipermetrin, alfa-metrin i heksakonazol) kojoj smo miševe izložili udisanjem i preko kože tijekom 6 sati dnevno i 5 dana tjedno do najviše 90 dana. Kronično izlaganje pesticidima vezano uz aplaziju koštane srži bilo je primarno potvrđeno krvnom slikom, celularnošću koštane srži, kratkotrajnom kulturom eksplantata koštane srži radi stanične kinetike, razmazom koštane srži i ekspresijskim obrascem protočne citometrije izvanstaničnog receptora Lin-Sca-1+C-kit+. Potom su analizirane sastavnice signalnog puta hedgehog u koštanoj srži kontrolnih jedinki i aplastičnih životinja koje su tretirane pesticidima. Rezultati su pokazali pancitopeniju periferne krvi, razvoju anomaliju neutrofi la, depresiju primitivnih matičnih stanica i prastanica uz Shh, Ptch1, Smo i Gli1 ekspresiju u skupini koja je imala aplaziju. Ovo istraživanje navodi na zaključak da pesticidi uzrokuju sniženje dvaju kritičnih proteina - Ptch1 i Gli1 - unutar hematopoetskih matičnih stanica i prastanica uzrokujući time hematopoetsku homeostazu i poremećaje regeneracijskog mehanizma in vivo zajedno s aplazijom koštane srži

Alteration in Marrow Stromal Microenvironment and Apoptosis Mechanisms Involved in Aplastic Anemia: An Animal Model to Study the Possible Disease Pathology

Aplastic anemia (AA) is a heterogeneous disorder of bone marrow failure syndrome. Suggested mechanisms include a primary stem cell deficiency or defect, a secondary stem cell defect due to abnormal regulation between cell death and differentiation, or a deficient microenvironment. In this study, we have tried to investigate the alterations in hematopoietic microenvironment and underlying mechanisms involved in such alterations in an animal model of drug induced AA. We presented the results of studying long term marrow culture, marrow ultra-structure, marrow adherent and hematopoietic progenitor cell colony formation, flowcytometric analysis of marrow stem and stromal progenitor populations and apoptosis mechanism involved in aplastic anemia. The AA marrow showed impairment in cellular proliferation and maturation and failed to generate a functional stromal microenvironment even after 19 days of culture. Ultra-structural analysis showed a degenerated and deformed marrow cellular association in AA. Colony forming units (CFUs) were also severely reduced in AA. Significantly decreased marrow stem and stromal progenitor population with subsequently increased expression levels of both the extracellular and intracellular apoptosis inducer markers in the AA marrow cells essentially pointed towards the defective hematopoiesis; moreover, a deficient and apoptotic microenvironment and the microenvironmental components might have played the important role in the possible pathogenesis of AA

Primitive Sca-1 Positive Bone Marrow HSC in Mouse Model of Aplastic Anemia: A Comparative Study through Flowcytometric Analysis and Scanning Electron Microscopy

Self-renewing Hematopoietic Stem Cells (HSCs) are responsible for reconstitution of all blood cell lineages. Sca-1 is the “stem cell antigen” marker used to identify the primitive murine HSC population, the expression of which decreases upon differentiation to other mature cell types. Sca-1+ HSCs maintain the bone marrow stem cell pool throughout the life. Aplastic anemia is a disease considered to involve primary stem cell deficiency and is characterized by severe pancytopenia and a decline in healthy blood cell generation system. Studies conducted in our laboratory revealed that the primitive Sca-1+ BM-HSCs (bone marrow hematopoietic stem cell) are significantly affected in experimental Aplastic animals pretreated with chemotherapeutic drugs (Busulfan and Cyclophosphamide) and there is increased Caspase-3 activity with consecutive high Annexin-V positivity leading to premature apoptosis in the bone marrow hematopoietic stem cell population in Aplastic condition. The Sca-1bright, that is, “more primitive” BM-HSC population was more affected than the “less primitive” BM-HSC Sca-1dim  population. The decreased cell population and the receptor expression were directly associated with an empty and deranged marrow microenvironment, which is evident from scanning electron microscopy (SEM). The above experimental evidences hint toward the manipulation of receptor expression for the benefit of cytotherapy by primitive stem cell population in Aplastic anemia cases

Single-cell characterization and metabolic profiling of in vitro cultured human skeletal progenitors with enhanced in vivo bone forming capacity.

Cell populations and their interplay provide the basis of a cell-based regenerative construct. Serum-free preconditioning can overcome the less predictable behavior of serum expanded progenitor cells, but the underlying mechanism and how this is reflected in vivo remains unknown. Herein, the cellular and molecular changes associated with a cellular phenotype shift induced by serum-free preconditioning of human periosteum-derived cells were investigated. Following BMP-2 stimulation, preconditioned cells displayed enhanced in vivo bone forming capacity, associated with an adapted cellular metabolism together with an elevated expression of BMPR2. Single-cell RNA sequencing confirmed the activation of pathways and transcriptional regulators involved in bone development and fracture healing, providing support for the augmentation of specified skeletal progenitor cell populations. The reported findings illustrate the importance of appropriate in vitro conditions for the in vivo outcome. In addition, BMPR2 represents a promising biomarker for the enrichment of skeletal progenitor cells for in vivo bone regeneration

Single-cell characterization and metabolic profiling of in vitro cultured human skeletal progenitors with enhanced in vivo bone forming capacity

Cell populations and their interplay provide the basis of a cell-based regenerative construct. Serum-free preconditioning can overcome the less predictable behavior of serum expanded progenitor cells, but the underlying mechanism and how this is reflected in vivo remains unknown. Herein, the cellular and molecular changes associated with a cellular phenotype shift induced by serum-free preconditioning of human periosteum-derived cells were investigated. Following BMP-2 stimulation, preconditioned cells displayed enhanced in vivo bone forming capacity, associated with an adapted cellular metabolism together with an elevated expression of BMPR2. Single-cell RNA sequencing confirmed the activation of pathways and transcriptional regulators involved in bone development and fracture healing, providing support for the augmentation of specified skeletal progenitor cell populations. The reported findings illustrate the importance of appropriate in vitro conditions for the in vivo outcome. In addition, BMPR2 represents a promising biomarker for the enrichment of skeletal progenitor cells for in vivo bone regeneration.status: publishe

Healing of a Large Long-Bone Defect through Serum-Free In Vitro Priming of Human Periosteum-Derived Cells

Clinical translation of cell-based strategies for regenerative medicine demands predictable in vivo performance where the use of sera during in vitro preparation inherently limits the efficacy and reproducibility. Here, we present a bioinspired approach by serum-free pre-conditioning of human periosteum-derived cells, followed by their assembly into microaggregates simultaneously primed with bone morphogenetic protein 2 (BMP-2). Pre-conditioning resulted in a more potent progenitor cell population, while aggregation induced osteochondrogenic differentiation, further enhanced by BMP-2 stimulation. Ectopic implantation displayed a cascade of events that closely resembled the natural endochondral process resulting in bone ossicle formation. Assessment in a critical size long-bone defect in immunodeficient mice demonstrated successful bridging of the defect within 4 weeks, with active contribution of the implanted cells. In short, the presented serum-free process represents a biomimetic strategy, resulting in a cartilage tissue intermediate that, upon implantation, robustly leads to the healing of a large long-bone defect.publisher: Elsevier articletitle: Healing of a Large Long-Bone Defect through Serum-Free In Vitro Priming of Human Periosteum-Derived Cells journaltitle: Stem Cell Reports articlelink: http://dx.doi.org/10.1016/j.stemcr.2017.01.005 content_type: article copyright: © 2017 The Author(s).status: publishe

Healing of a Large Long-Bone Defect through Serum-Free In Vitro Priming of Human Periosteum-Derived Cells

Clinical translation of cell-based strategies for regenerative medicine demands predictable in vivo performance where the use of sera during in vitro preparation inherently limits the efficacy and reproducibility. Here, we present a bioinspired approach by serum-free pre-conditioning of human periosteum-derived cells, followed by their assembly into microaggregates simultaneously primed with bone morphogenetic protein 2 (BMP-2). Pre-conditioning resulted in a more potent progenitor cell population, while aggregation induced osteochondrogenic differentiation, further enhanced by BMP-2 stimulation. Ectopic implantation displayed a cascade of events that closely resembled the natural endochondral process resulting in bone ossicle formation. Assessment in a critical size long-bone defect in immunodeficient mice demonstrated successful bridging of the defect within 4 weeks, with active contribution of the implanted cells. In short, the presented serum-free process represents a biomimetic strategy, resulting in a cartilage tissue intermediate that, upon implantation, robustly leads to the healing of a large long-bone defect