Activity Of Progenitor Cells Research Articles

Abstract P1-10-06: Effect of aging and mTOR inhibition on human mammary stem cells and early breast cancer progression markers

Abstract Ductal carcinoma in situ (DCIS) is the most common form of breast cancer and is a predecessor to invasive carcinoma development. The origins for early breast cancer have been attributed to aberrant single cells, which may include adult stem and progenitor cells. We performed a window trial with mTOR inhibitor sirolimus in patients with atypical ductal hyperplasia (ADH) or DCIS and investigated the epithelial populations in adjacent normal and ADH/DCIS breast tissues along with their residing stem and progenitor cell activity. Differences associated with age, menopausal status, and treatment by sirolimus have also been explored. While the mammary epithelial populations were seen to be unchanged in aging, we found that age correlates with a decline in stem cell activity. mTOR inhibition was also able to abrogate mammary stem cell activity and decrease luminal progenitor passaging lifespan, particularly in postmenopausal patients. While mTOR inhibition had no effect on myoepithelial populations, it did decrease premenopausal luminal cell populations and reduced prognostic markers associated with breast cancer progression from DCIS to invasive breast cancer. These markers include p16INK4A, COX-2, and Ki67, which were reduced in DCIS breast tissue from patients administered with sirolimus as shown through quantitative analysis of immunohistochemistry. Overall, these findings indicate a link from mTOR signaling to mammary stem cell activity and cancer progression. mTOR inhibitors may have potential for breast cancer prevention. Citation Format: Larry E Broome, Hakim Bouamar, Kate Lathrop, Ismail Jatoi, Andrew Brenner, Michael Garcia, Alia Nazarullah, Karla Moncada, Virginia Kaklamani, Lu-Zhe Sun. Effect of aging and mTOR inhibition on human mammary stem cells and early breast cancer progression markers [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-10-06.

Telomerase is required for glomerular renewal in kidneys of adult mice

Homeostatic renal filtration relies on the integrity of podocytes, which function in glomerular filtration. These highly specialized cells are damaged in 90% of chronic kidney disease, representing the leading cause of end-stage renal failure. Although modest podocyte renewal has been documented in adult mice, the mechanisms regulating this process remain largely unknown and controversial. Using a mouse model of Adriamycin-induced nephropathy, we find that the recovery of filtration function requires up-regulation of the endogenous telomerase component TERT. Previous work has shown that transient overexpression of catalytically inactive TERT (i-TERTci mouse model) has an unexpected role in triggering dramatic podocyte proliferation and renewal. We therefore used this model to conduct specific and stochastic lineage-tracing strategies in combination with high throughput sequencing methods. These experiments provide evidence that TERT drives the activation and clonal expansion of podocyte progenitor cells. Our findings demonstrate that the adult kidney bears intrinsic regenerative capabilities involving the protein component of telomerase, paving the way for innovative research toward the development of chronic kidney disease therapeutics.

Disrupted Cacna1c gene expression perturbs spontaneous Ca2+ activity causing abnormal brain development and increased anxiety

The L-type voltage-gated Ca2+ channel gene CACNA1C is a risk gene for various psychiatric conditions, including schizophrenia and bipolar disorder. However, the cellular mechanism by which CACNA1C contributes to psychiatric disorders has not been elucidated. Here, we report that the embryonic deletion of Cacna1c in neurons destined for the cerebral cortex using an Emx1-Cre strategy disturbs spontaneous Ca2+ activity and causes abnormal brain development and anxiety. By combining computational modeling with electrophysiological membrane potential manipulation, we found that neural network activity was driven by intrinsic spontaneous Ca2+ activity in distinct progenitor cells expressing marginally increased levels of voltage-gated Ca2+ channels. MRI examination of the Cacna1c knockout mouse brains revealed volumetric differences in the neocortex, hippocampus, and periaqueductal gray. These results suggest that Cacna1c acts as a molecular switch and that its disruption during embryogenesis can perturb Ca2+ handling and neural development, which may increase susceptibility to psychiatric disease.

Safe and Effective Cynomolgus Monkey GLP-Tox Study with Repetitive Intrathecal Application of a TGFBR2 Targeting LNA-Gapmer Antisense Oligonucleotide as Treatment Candidate for Neurodegenerative Disorders.

The capability of the adult central nervous system to self-repair/regenerate was demonstrated repeatedly throughout the last decades but remains in debate. Reduced neurogenic niche activity paralleled by a profound neuronal loss represents fundamental hallmarks in the disease course of neurodegenerative disorders. We and others have demonstrated the endogenous TGFβ system to represent a potential pathogenic participant in disease progression, of amyotrophic lateral sclerosis (ALS) in particular, by generating and promoting a disequilibrium of neurodegenerative and neuroregenerative processes. The novel human/primate specific LNA Gapmer Antisense Oligonucleotide “NVP-13”, targeting TGFBR2, effectively reduced its expression and lowered TGFβ signal transduction in vitro and in vivo, paralleled by boosting neurogenic niche activity in human neuronal progenitor cells and nonhuman primate central nervous system. Here, we investigated NVP-13 in vivo pharmacology, safety, and tolerability following repeated intrathecal injections in nonhuman primate cynomolgus monkeys for 13 weeks in a GLP-toxicology study approach. NVP-13 was administered intrathecally with 1, 2, or 4 mg NVP-13/animal within 3 months on days 1, 15, 29, 43, 57, 71, and 85 in the initial 13 weeks. We were able to demonstrate an excellent local and systemic tolerability, and no adverse events in physiological, hematological, clinical chemistry, and microscopic findings in female and male Cynomolgus Monkeys. Under the conditions of this study, the no observed adverse effect level (NOAEL) is at least 4 mg/animal NVP-13.

Identification and Validation of CRISPR/Cas9 Off-Target Activity in Hematopoietic Stem and Progenitor Cells.

Targeted genome editing in hematopoietic stem and progenitor cells (HSPCs) using CRISPR/Cas9 can potentially provide a permanent cure for hematologic diseases. However, the utility of CRISPR/Cas9 systems for therapeutic genome editing can be compromised by their off-target effects. In this chapter, we outline the procedures for CRISPR/Cas9 off-target identification and validation in HSPCs. This method is broadly applicable to diverse CRISPR/Cas9 systems and cell types. Using this protocol, researchers can perform computational prediction and experimental identification of potential off-target sites followed by off-target activity quantification by next-generation sequencing.

ANALYSIS OF THE INFLUENCE OF PROLONGED IRRADIATION ON HEMATOPOIETIC PROGENITOR CELLS IN GEL DIFFUSION CHAMBERS USING MATHEMATICAL MODELLING.

determining of the functional activity of mice bone marrow hematopoietic progenitor cells, cultivated in gel diffusion chambers, on the stages of hematopoiesis recovery after their prolonged irradiation in the lethal dose in a comparative aspect with the method of colony forming in spleen using mathematical model. The method of cell cultivation in gel diffusion chambers, cytological methods, mathematical modeling, and statistical methods of research were used. Bone marrow samples extracted from the femur of mice irradiated with a total dose of 8 Gy with a power 0.0028 Gy/min were cultivated in diffusion chambers with semi solid agar in the abdominal cavity of CBA recipient mice. Comparative analysis of the colonyforming efficiency of progenitor cells (CFU) was carried out during cultivation in gel diffusion chambers in the process of hematopoiesis recovery for 30 days, as well as in the spleen of lethally irradiated animals, in accordance with the mathematical model. Analysis of colony forming kinetics in gel diffusion chambers after prolonged exposure to ionizing radiation indicated the biphasic nature of hematopoiesis recovery. Thus, in the first few days after the irradiation a drop in the number of CFU is observed compared to the control, which continues until the 9th day. Subsequently there is a sharp increase in the number of CFU in cell culture, which continues until the complete recovery of hematopoiesis. The obtained data, recalculated per mouse femur, correspond to the results of colony forming in the spleen of irradiated animals, described by K. S. Chertkov and taken as a basis while developing our mathematical model, as well as to its parameters, which describe the process of hematopoiesis recovery. Conformity of the indices obtained during the cultivation using the method of gel diffusion chambers of mice bone marrow prolongedly irradiated at a total dose of 8 Gy with a power 0.0028 Gy/min, to the results of colony forming in spleen of lethally irradiated mice, which were the basis for mathematical model development, is the evidence of the feasibility of using a mathematical model to assess the process of hematopoiesis recovery by progenitor cells of different maturation levels, and the experimental approach of CFU growing in gel diffusion chambers can be considered as an additional method of researching the hematopoiesis recovery along with the spleen colony method.

Both Vitamin D Supplementation and HIIT Boost Muscle VDR Expression, Which May Underlie Benefits for Frailty

Frailty is a condition of poor response to stressors that increases susceptibility to adverse outcomes - including disability and death. Declining physical function is an important hallmark of frailty, and we previously published that long term vitamin D insufficiency from young to middle-age leads to declines in endurance and gait disturbances. Furthermore, we report that aged mice (24-months) made vitamin D insufficient for 4 months exhibit increased frailty, whereas those made hyper-sufficient do not. Exercise, including short session high intensity interval training (HIIT - 10 minutes/3x-week), also reverses frailty in aged mice. Here we investigate the impacts of aging, vitamin D, and exercise on underlying muscle quality and muscle stem cell activity. Our preliminary data reveal muscle vitamin D receptor (VDR) expression is lower in aged mice (24-28 months) relative to young mice (6 months). Yet HIIT, either one hour after a single session or following 6 weeks, increases VDR expression. HIIT also increases myonuclear accretion in muscle fibers – an indicator of in vivo stem cell activity – and stimulates progenitor cells proliferation ex vivo. Likewise, we observe that vitamin D hyper supplementation alone also increases muscle VDR expression and the number of satellite cells. These data indicate that both vitamin D supplementation and HIIT independently enhance VDR expression in skeletal muscle with associated greater satellite and muscle progenitor cell activity. These data critically link vitamin D physiology and HIIT in muscle, and thus provide a mechanistic basis for their benefits for muscle quality, function, and health during aging.

Regulation of neurogenesis and cerebral angiogenesis by cell protein proteolysis products

Brain development is a unique process characterized by mechanisms defined as neuroplasticity (synaptogenesis, synapse elimination, neurogenesis, and cerebral angiogenesis). Numerous neurodevelopmental disorders brain damage, and aging are manifested by neurological deficits that are caused by aberrant neuroplasticity. The presence of stem and progenitor cells in neurogenic niches of the brain is responsible for the formation of new neurons capable of integrating into preexisting synaptic assemblies. The determining factors for the cells within the neurogenic niche are the activity of the vascular scaffold and the availability of active regulatory molecules that establish the optimal microenvironment. It has been found that regulated intramembrane proteolysis plays an important role in the control of neurogenesis in brain neurogenic niches. Molecules generated by the activity of specific proteases can stimulate or suppress the activity of neural stem and progenitor cells, their proliferation and differentiation, migration and integration of newly formed neurons into synaptic networks. Local neoangiogenesis supports the processes of neurogenesis in neurogenic niches, which is guaranteed by the multivalent action of peptides formed from transmembrane proteins. Identification of new molecules regulating the neuroplasticity (neurogenesis and angiogenesis). i. e. enzymes, substrates, and products of intramembrane proteolysis, will ensure the development of protocols for detecting the neuroplasticity markers and targets for efficient pharmacological modulation.

Phase Separation Mediates NUP98 Fusion Oncoprotein Leukemic Transformation.

We show that homotypic and heterotypic mechanisms of LLPS control NUP98-HOXA9 puncta formation, modulating transcriptional activity and transforming hematopoietic cells. Importantly, these mechanisms are generalizable to other NUP98 FOs that share similar domain structures. These findings address long-standing questions on how nuclear puncta form and their link to leukemogenesis. This article is highlighted in the In This Issue feature, p. 873.

EPCR Signaling Controls the Activity of Hematopoietic Stem Cells Independent of Coagulation Regulation

BackgroundAll mature blood cells are derived from multipotent hematopoietic stem cells (HSCs) which are activated to meet the demand of the host during inflammation and injury. The endothelial cell protein C receptor (EPCR) is a marker for primitivity and quiescence of HSCs but the relative contributions of EPCR signaling versus anticoagulant functions in HSC maintenance are incompletely defined.AimsWe aimed to dissect functions of EPCR by studying anticoagulant and signaling function in HSC of EPCR C/S mice carrying a single intracellular point mutation abolishing normal trafficking of EPCR through endo-lysosomal compartments. We assessed the contributions of EPCR signaling to stem cell maintenance by analyzing HSC mobilization and leukemia progression.MethodsWe studied the frequency and cell cycle activity of bone marrow (BM) hematopoietic stem and progenitor cells (HSPC) by multicolor flow cytometry. Furthermore, we analyzed changes in hematopoiesis in steady state, after granulocyte colony stimulating factor (G-CSF)-induced mobilization, in the context of aging and in the context of leukemia, using the MLL-AF9-induced acute myeloid leukemia (AML) model.ResultsHSCs, lungs and isolated lung-derived smooth muscle cells of EPCR C/S mice showed protein expression levels and anticoagulant function indistinguishable from wildtype (WT). We found an increase of circulating HSCs in the peripheral blood of EPCR C/S mice compared to control under steady state conditions. Isolated HSC displayed diminished polarization of CDC42 and VLA-4 (α 4β 1 integrin) affinity to VCAM-1 in EPCR C/S versus strain-matched EPCR wt mice, indicating that EPCR signaling directly controls HSC retention via integrin affinity to the BM niche. In addition, we noticed a higher cell cycle activity in myeloid-restricted progenitors of EPCR C/S mice compared to control. G-CSF treatment led to increased mobilization of both BM neutrophils and HSCs into the peripheral blood of EPCR C/S mice compared to EPCR wt mice. A myeloid bias was also seen in serially transplanted aged mice, resulting in increased frequencies of myeloid-biased progenitors in the BM of EPCR C/S mice compared to control mice, accompanied by an increase of circulating neutrophils in the blood. Consistent with higher cell cycle activity of myeloid progenitors and an overall increase of myeloid-biased output in EPCR C/S mice, induction of AML by retroviral transduction of EPCR C/S BM cells with MLL-AF9-expressing retrovirus resulted in an increase of cell cycle activity of Lin - MLL-AF9 + leukemic BM blasts and a higher leukemic load in the peripheral blood of mice transplanted with MLL-AF9 + EPCR C/S BM compared to control. As a result, MLL-AF9 + EPCR C/S leukemia showed a more aggressive disease with shortened survival times compared to control. In contrast, chemotherapy of MLL-AF9 + EPCR C/S leukemia reduced leukemic load in the peripheral blood and decelerated disease progression. These data demonstrate that increased leukemia cell cycle activity conferred chemosensitivity.ConclusionWith a site-specific EPCR mutant knock-in mouse, we here demonstrate that EPCR signaling and anticoagulant function can be separated. We provide direct evidence that EPCR signaling plays a crucial role in maintaining HSC retention via VLA-4 affinity to VCAM-1, controls cell cycle activity and myeloid output in normal, stress-induced, and malignant hematopoiesis with implications for therapeutic approaches in acute myeloid leukemia. DisclosuresRuf: MeruVasimmune: Other: Ownership Interest; ARCA bioscience: Consultancy, Patents & Royalties; ICONIC Therapeutics: Consultancy.

ВЛИЯНИЕ МОЩНОГО НЕИОНИЗИРУЮЩЕГО ТЕРАГЕРЦОВОГО ИЗЛУЧЕНИЯ НА ЗДОРОВЫЕ И ОПУХОЛЕВЫЕ КЛЕТКИ ЧЕЛОВЕКА НЕЙРАЛЬНОГО ПРОИСХОЖДЕНИЯ

Purpose: Study of the influence of high-power pulses of coherent non-ionizing terahertz (THz) radiation on the formation of foci of double-strand DNA breaks and the proliferative activity of human neuronal cells.
 Material and methods: Irradiated cell cultures are direct reprogramming neural progenitor cells (drNPCs), neuroblastoma cells (SK-N-BE). Cells are irradiated with a sequence of THz radiation pulses with a peak intensity of ~ 20 GW/cm2 and electric field strength of 2.8 MV/cm. Irradiation lasts 30 mins.
 Results: There is no statistically significant difference in the number of γH2AX histone foci between experimental and control cell groups.
 Conclusion: It was shown that a short exposure (30 min) of cells to THz radiation with intensity of 20 GW/cm2 does not affect the proliferative activity of both neural progenitor cells and neuroblastoma cells and does not cause a significant increase in γH2AX foci in any of the studied cell lines.

Analysis of the early response to spinal cord injury identified a key role for mTORC1 signaling in the activation of neural stem progenitor cells

Xenopus laevis are able to regenerate the spinal cord during larvae stages through the activation of neural stem progenitor cells (NSPCs). Here we use high-resolution expression profiling to characterize the early transcriptome changes induced after spinal cord injury, aiming to identify the signals that trigger NSPC proliferation. The analysis delineates a pathway that starts with a rapid and transitory activation of immediate early genes, followed by migration processes and immune response genes, the pervasive increase of NSPC-specific ribosome biogenesis factors, and genes involved in stem cell proliferation. Western blot and immunofluorescence analysis showed that mTORC1 is rapidly and transiently activated after SCI, and its pharmacological inhibition impairs spinal cord regeneration and proliferation of NSPC through the downregulation of genes involved in the G1/S transition of cell cycle, with a strong effect on PCNA. We propose that the mTOR signaling pathway is a key player in the activation of NPSCs during the early steps of spinal cord regeneration.

Oncostatin M promotes hepatic progenitor cell activation and hepatocarcinogenesis via macrophage-derived tumor necrosis factor-α

Hepatocellular carcinoma (HCC) usually occurs at the late stage of chronic liver injury. Oncostatin M (OSM) is a tumor-associated cytokine highly expressed in cirrhosis and HCC patients; however, its role in hepatocarcinogenesis has not been clearly elucidated. In this study, we investigated the effect of OSM on HCC occurrence in a rat model of N-diethylnitrosamine-induced HCC. OSM overexpression significantly increased the number of tumor nodules and shortened the overall survival of the rats. Notably, OSM promoted HPC activation in vivo but did not directly regulate the proliferation of the HPC cell line in vitro. Further, OSM induced tumor necrosis factor-α (TNF-α) secretion and CD68+ macrophage accumulation, which were positively correlated with HPC activation. Additionally, TNF-α or macrophage depletion inhibited the promoting effect of OSM on hepatocarcinogenesis and HPC activation. Furthermore, OSM expression in the peritumoral tissues of HCC was positively correlated with poor overall survival of patients. In conclusion, OSM plays an important role in hepatocarcinogenesis by regulating the liver inflammation environment. Hence, OSM could be used as a potential target for HCC prevention and therapy or as an indicator of HCC prognosis.

The protease-inhibitor SerpinB3 as a critical modulator of the stem-like subset in human cholangiocarcinoma.

Background and aimsCholangiocarcinoma (CCA) is a form of primary liver cancer with limited therapeutic options. Recently, cancer stem cells (CSCs) have been proposed as a driving force of tumour initiation and dissemination, thus representing a crucial therapeutic target. The protease inhibitor SerpinB3 (SB3) has been identified in several malignancies including hepatocellular carcinoma. SB3 has been involved in the early events of hepatocarcinogenesis and is highly expressed in hepatic progenitor cells and in a mouse model of liver progenitor cell activation. However, only limited information on the possible role of SB3 in CCA stem‐like compartment is available.MethodsEnrichment of CCA stem‐like subset was performed by sphere culture (SPH) in CCA cell lines (CCLP1, HUCCT1, MTCHC01 and SG231). Quantitative RT‐PCR and Western blotting were used to detect SB3 in both SPH and parental monolayer (MON) cells. Acquired CSC‐like features were analysed using an endogenous and a paracrine in vitro model, with transfection of SB3 gene or addition of recombinant SB3 to cell medium respectively. SB3 tumorigenic role was explored in an in vivo mouse model of CCA by subcutaneous injection of SB3‐transfected MON (MONSB3+) cells in immune‐deficient NOD‐SCID/IL2Rg null (NSG) mice. SB3 expression in human CCA sections was investigated by immunohistochemistry. Overall survival (OS) and time to recurrence (TTR) analyses were carried out from a transcriptome database of 104 CCA patients.ResultsSB3, barely detected in parental MON cells, was overexpressed in the same CCA cells grown as 3D SPH. Notably, MONSB3+ showed significant overexpression of genes associated with stemness (CD24, CD44, CD133), pluripotency (c‐MYC, NOTCH1, STAT3, YAP, NANOG, BMI1, KLF4, OCT4, SOX2), epithelial mesenchymal transition (β‐catenin, SLUG) and extracellular matrix remodelling (MMP1, MMP7, MMP9, ADAM9, ADAM10, ADAM17, ITGB3). SB3‐overexpressing cells showed superior spherogenic capacity and invasion ability compared to control. Importantly, MONSB3+ exhibited activation of MAP kinases (ERK1/2, p38, JNK) as well as phosphorylation of NFκB (p65) in addition to up‐regulation of the proto‐oncogene β‐catenin. All these effects were reversed after transient silencing of SB3. According to the in vitro finding, MONSB3+ cells retained high tumorigenic potential in NSG mice. SB3 overexpression was observed in human CCA tissues and analysis of OS as well as TTR indicated a worse prognosis in SB3+ CCA patients.ConclusionThese findings indicate a SB3 role in mediating malignant phenotype of CCA and identify a new therapeutic target.

Deferoxamine accelerates endothelial progenitor cell senescence and compromises angiogenesis.

Senescence reduces the circulating number and angiogenic activity of endothelial progenitor cells (EPCs), and is associated with aging-related vascular diseases. However, it is very time-consuming to obtain aged cells (~1 month of repeated replication) or animals (~2 years) for senescence studies. Here, we established an accelerated senescence model by treating EPCs with deferoxamine (DFO), an FDA-approved iron chelator. Four days of low-dose (3 μM) DFO induced senescent phenotypes in EPCs, including a senescent pattern of protein expression, impaired mitochondrial bioenergetics, altered mitochondrial protein levels and compromised angiogenic activity. DFO-treated early EPCs from young and old donors (< 35 vs. > 70 years old) displayed similar senescent phenotypes, including elevated senescence-associated β-galactosidase activity and reduced relative telomere lengths, colony-forming units and adenosine triphosphate levels. To validate this accelerated senescence model in vivo, we intraperitoneally injected Sprague-Dawley rats with DFO for 4 weeks. Early EPCs from DFO-treated rats displayed profoundly senescent phenotypes compared to those from control rats. Additionally, in hind-limb ischemic mice, DFO pretreatment compromised EPC angiogenesis by reducing both blood perfusion and capillary density. DFO thus accelerates EPC senescence and appears to hasten model development for cellular senescence studies.

Lymphoid-biased hematopoietic stem cells and myeloid-biased hematopoietic progenitor cells have radioprotection activity.

Radioprotection was previously considered as a function of hematopoietic stem cells (HSCs). However, recent studies have reported its activity in hematopoietic progenitor cells (HPCs). To address this issue, we compared the radioprotection activity in 2 subsets of HSCs (nHSC1 and 2 populations) and 4 subsets of HPCs (nHPC1–4 populations) of the mouse bone marrow, in relation to their in vitro and in vivo colony-forming activity. Significant radioprotection activity was detected in the nHSC2 population enriched in lymphoid-biased HSCs. Moderate radioprotection activity was detected in nHPC1 and 2 populations enriched in myeloid-biased HPCs. Low radioprotection activity was detected in the nHSC1 enriched in myeloid-biased HSCs. No radioprotection activity was detected in the nHPC3 and 4 populations that included MPP4 (LMPP). Single-cell colony assay combined with flow cytometry analysis showed that the nHSC1, nHSC2, nHPC1, and nHPC2 populations had the neutrophils/macrophages/erythroblasts/megakaryocytes (nmEMk) differentiation potential whereas the nHPC3 and 4 populations had only the nm differentiation potential. Varying day 12 spleen colony-forming units (day 12 CFU-S) were detected in the nHSC1, nHSC2, and nHPC1–3 populations, but very few in the nHPC4 population. These data suggested that nmEMk differentiation potential and day 12 CFU-S activity are partially associated with radioprotection activity. Reconstitution analysis showed that sufficient myeloid reconstitution around 12 to 14 days after transplantation was critical for radioprotection. This study implied that radioprotection is specific to neither HSC nor HPC populations, and that lymphoid-biased HSCs and myeloid-biased HPCs as populations play a major role in radioprotection.

Type V collagen regulates the structure and biomechanics of TMJ condylar cartilage: A fibrous-hyaline hybrid

This study queried the role of type V collagen in the post-natal growth of temporomandibular joint (TMJ) condylar cartilage, a hybrid tissue with a fibrocartilage layer covering a secondary hyaline cartilage layer. Integrating outcomes from histology, immunofluorescence imaging, electron microscopy and atomic force microscopy-based nanomechanical tests, we elucidated the impact of type V collagen reduction on TMJ condylar cartilage growth in the type V collagen haploinsufficiency and inducible knockout mice. Reduction of type V collagen led to significantly thickened collagen fibrils, decreased tissue modulus, reduced cell density and aberrant cell clustering in both the fibrous and hyaline layers. Post-natal growth of condylar cartilage involves the chondrogenesis of progenitor cells residing in the fibrous layer, which gives rise to the secondary hyaline layer. Loss of type V collagen resulted in reduced proliferation of these cells, suggesting a possible role of type V collagen in mediating the progenitor cell niche. When the knockout of type V collagen was induced in post-weaning mice after the start of physiologic TMJ loading, the hyaline layer exhibited pronounced thinning, supporting an interplay between type V collagen and occlusal loading in condylar cartilage growth. The phenotype in hyaline layer can thus be attributed to the impact of type V collagen on the mechanically regulated progenitor cell activities. In contrast, knee cartilage does not contain the progenitor cell population at post-natal stages, and develops normal structure and biomechanical properties with the loss of type V collagen. Therefore, in the TMJ, in addition to its established role in regulating the assembly of collagen I fibrils, type V collagen also impacts the mechanoregulation of progenitor cell activities in the fibrous layer. We expect such knowledge to establish a foundation for understanding condylar cartilage matrix development and regeneration, and to yield new insights into the TMJ symptoms in patients with classic Ehlers-Danlos syndrome, a genetic disease due to autosomal mutation of type V collagen.

An insight on the impact of teleost whole genome duplication on the regulation of the molecular networks controlling skeletal muscle growth.

Fish muscle growth is a complex process regulated by multiple pathways, resulting on the net accumulation of proteins and the activation of myogenic progenitor cells. Around 350–320 million years ago, teleost fish went through a specific whole genome duplication (WGD) that expanded the existent gene repertoire. Duplicated genes can be retained by different molecular mechanisms such as subfunctionalization, neofunctionalization or redundancy, each one with different functional implications. While the great majority of ohnolog genes have been identified in the teleost genomes, the effect of gene duplication in the fish physiology is still not well characterized. In the present study we studied the effect of WGD on the transcription of the duplicated components controlling muscle growth. We compared the expression of lineage-specific ohnologs related to myogenesis and protein balance in the fast-skeletal muscle of pacus (Piaractus mesopotamicus—Ostariophysi) and Nile tilapias (Oreochromis niloticus—Acanthopterygii) fasted for 4 days and refed for 3 days. We studied the expression of 20 ohnologs and found that in the great majority of cases, duplicated genes had similar expression profiles in response to fasting and refeeding, indicating that their functions during growth have been conserved during the period after the WGD. Our results suggest that redundancy might play a more important role in the retention of ohnologs of regulatory pathways than initially thought. Also, comparison to non-duplicated orthologs showed that it might not be uncommon for the duplicated genes to gain or loss new regulatory elements simultaneously. Overall, several of duplicated ohnologs have similar transcription profiles in response to pro-growth signals suggesting that evolution tends to conserve ohnolog regulation during muscle development and that in the majority of ohnologs related to muscle growth their functions might be very similar.

Creating a Pro-Regenerative Tissue Microenvironment: Local Control is the Key.

Creating a Pro-Regenerative Tissue Microenvironment: Local Control is the Key.

The ability of bone marrow progenitor cells to form colonies in ex vivo culture of MDS patients

The results of in vitro hematopoiesis studies have provided most of the knowledge about the organization, regulation, and development of the human hematopoietic system over the past three to four decades. However, due to the impossibility of an appropriate assessment of hematopoietic stem cells (HSC) in humans and because of the shortcomings of methodological approaches to determining the role of hematopoietic progenitor cells in the pathogenesis of MDS and to predicting the course of the pathological process, semiliquid agar cultures of bone marrow from patients with myelodysplastic syndrome were used. Myelodysplastic syndrome (MDS) refers to a clinically, morphologically, and genetically heterogeneous group of diseases characterized by clonalism and arising from mutations at the level of hematopoietic progenitor cells. Proliferation of such a mutated stem cell progenitors leads to ineffective maturation of myeloid lineage cells and dysplastic changes in the bone marrow (BM). The aim of the study was to establish the relationship between the functional activity of hematopoietic progenitor cells in the ex vivo culture and the activity of the pathological process in the myelodysplastic syndrome. We studied bone marrow samples from patients with the myelodysplastic syndrome, namely refractory anemia with excess blasts I (MDS RAEB I) and refractory anemia with excess blasts II (MDS RAEB II) and AML under conditions in vitro, as well as their clinical laboratory data. It was found that the percentage of blasts and myeloblasts in the samples of patients with AML and MDS RAEB II increased, compared to the samples of patients with MDS RAEB I (63.5±3.9 %, 18.05±1.01 % and 9.49±1.53 % respectively). An increase in the number of erythrocytes and hemoglobin content was noted in the group of patients with MDS RAEB I compared with MDS RAEB II (2.9±1.4×1012 / l and 105.04±3.6 g / l versus 9±0.8×1012 / l and 84.5±4.8 g / l, respectively). The analysis of the results of BM studies of patients with MDS in in vitro culture indicated a significant lag in the formation of cell aggregates during cultivation and a pronounced inhibition of the colony-forming ability of progenitor cells, compared to the control. A noticeable decrease in the colony-forming ability was observed in patients with MDS RAEB I, MDS RAEB II and AML in this sequence – 4.1±1.2 per 1×105 explanted cells, 3.2±0.9 per 1×105 explanted cells and 2.0±0.6 per 1×105 explanted cells, respectively. The analysis of hematological parameters and the results of BM cells cultivation at different stages of MDS indicates that the colony-forming ability of progenitor cells correlates with the depth of the pathological process.