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Thudium C.S.,Nordic Bioscience A S | Thudium C.S.,Lund Strategic Center for Stem Cell Biology | Moscatelli I.,Lund Strategic Center for Stem Cell Biology | Flores C.,Lund Strategic Center for Stem Cell Biology | And 7 more authors.
Calcified Tissue International | Year: 2014

Osteopetrosis due to lack of acid secretion by osteoclasts is characterized by abolished bone resorption, increased osteoclast numbers, but normal or even increased bone formation. In contrast, osteoclast-poor osteopetrosis appears to have less osteoblasts and reduced bone formation, indicating that osteoclasts are important for regulating osteoblast activity. To illuminate the role of the osteoclast in controlling bone remodeling, we transplanted irradiated skeletally mature 3-month old wild-type mice with hematopoietic stem cells (HSCs) to generate either an osteoclast-rich or osteoclast-poor adult osteopetrosis model. We used fetal liver HSCs from (1) oc/oc mice, (2) RANK KO mice, and (3) compared these to wt control cells. TRAP5b activity, a marker of osteoclast number and size, was increased in the oc/oc recipients, while a significant reduction was seen in the RANK KO recipients. In contrast, the bone resorption marker CTX-I was similarly decreased in both groups. Both oc/oc and Rank KO recipients developed a mild osteopetrotic phenotype. However, the osteoclast-rich oc/oc recipients showed higher trabecular bone volume (40 %), increased bone strength (66 %), and increased bone formation rate (54 %) in trabecular bone, while RANK KO recipients showed only minor trends compared to control recipients. We here show that maintaining non-resorbing osteoclasts, as opposed to reducing the osteoclasts, leads to increased bone formation, bone volume, and ultimately higher bone strength in vivo, which indicates that osteoclasts are sources of anabolic molecules for the osteoblasts. © 2014 Springer Science+Business Media.


Flores C.,Lund Strategic Center for Stem Cell Biology | Moscatelli I.,Lund Strategic Center for Stem Cell Biology | Thudium C.S.,Lund Strategic Center for Stem Cell Biology | Gudmann N.S.,Nordic Bioscience | And 5 more authors.
Haematologica | Year: 2013

The osteoclast is vital for establishment of normal hematopoiesis in the developing animal. However, its role for maintenance of hematopoiesis in adulthood is more controversial. To shed more light on this process, we transplanted hematopoietic stem cells from two osteopetrotic mouse models, with lack of osteoclasts or defective osteoclast function, to normal adult mice and examined the bone phenotype and hematopoiesis in the recipients. B6SJL mice were lethally irradiated and subsequently transplanted with oc/oc, Receptor Activator of Nuclear Factor Kappa B knockout or control fetal liver cells. Osteoclasts derived from the recipient animals were tested in vitro for osteoclas-togenesis and resorptive function. Bone remodeling changes were assessed using biomarkers of bone turnover and micro-CT. Hematopoiesis was assessed by flow cytometry and colony formation, and hematopoietic stem cell function by secondary competitive transplantations and cell cycle analysis. After transplantation, a donor chimerism of 97-98% was obtained, and by 15 weeks mild osteopetrosis had developed in recipients of cells from osteopetrotic mice. There were no alterations in the number of bone marrow cells. Colony formation was slightly reduced in Receptor Activator of Nuclear Factor Kappa B knock-out recipients but unchanged in oc/oc recipients. Phenotypically, stem cells were marginally reduced in recipients of cells from osteopetrotic mice, but no significant difference was seen in cell cycle status and in competitive secondary transplantations all three groups performed equally well. Our results indicate that osteoclast function is not crucial for hematopoietic stem cell maintenance in adult mice. © 2013 Ferrata Storti Foundation.


Lukin K.,Health Integrated | Fields S.,Health Integrated | Guerrettaz L.,Health Integrated | Straign D.,Health Integrated | And 7 more authors.
European Journal of Immunology | Year: 2011

In the absence of early B-cell factor 1 (EBF1), B-cell development is arrested at an uncommitted progenitor stage that exhibits increased lineage potentials. Previously, we investigated the roles of EBF1 and its DNA-binding partner Runx1 by evaluating B lymphopoiesis in single (EBF1het and Runx1het) and compound haploinsufficent (Ebf1+/-Runx1+/-, ERhet) mice. Here, we demonstrate that decreased Ebf1 gene dosage results in the inappropriate expression of NK-cell lineage-specific genes in B-cell progenitors. Moreover, prolonged expression of Ly6a/Sca-1 suggested the maintenance of a relatively undifferentiated phenotype. These effects were exacerbated by reduced expression of Runx1 and occurred despite expression of Pax5. Repression of inappropriately expressed genes was restored in most pre-B and all immature B cells of ERhet mice. Enforced EBF1 expression repressed promiscuous transcription in pro-B cells of ERhet mice and in Ebf1-/-Pax5-/- fetal liver cells. Together, our studies suggest that normal levels of EBF1 are critical for maintaining B-cell identity by directing repression of non-B-cell-specific genes. © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.


Henriksen K.,Nordic Bioscience A S | Andreassen K.V.,Nordic Bioscience A S | Thudium C.S.,Nordic Bioscience A S | Thudium C.S.,Lund Strategic Center for Stem Cell Biology | And 8 more authors.
Bone | Year: 2012

Osteoclasts are known to be important for the coupling process between bone resorption and formation. The aim of this study was to address when osteoclasts are anabolically active. Human monocytes were differentiated into mature osteoclasts by treatment with M-CSF and RANKL. Conditioned medium was collected from macrophages, pre-osteoclasts, and mature functional or non-resorbing osteopetrotic osteoclasts on either bone, plastic, decalcified bone or dentine with or without diphyllin, E64 or GM6001. Osteoclasts numbers were measured by TRACP activity. Bone resorption was evaluated by CTX-I and calcium release. The osteoblastic cell line 2. T3 was treated with 50% of CM or non-CM for 12. days. Bone formation was assessed by Alizarin Red extraction. CM from mature osteoclasts induced bone formation, while CM from macrophages did not. Non-resorbing osteoclasts generated from osteopetrosis patients showed little resorption, but still an induction of bone formation by osteoblasts. Mimicking the reduction in bone resorption using the V-ATPase inhibitor Diphyllin, the cysteine proteinase inhibitor E64 and the MMP-inhibitor GM6001 showed that CM from diphyllin and E64 treated osteoclasts showed reduced ability to induce bone formation compared to CM from vehicle treated osteoclasts, while CM from GM6001 treated osteoclasts equaled vehicle CM. Osteoclasts on either dentine or decalcified bone showed strongly attenuated anabolic capacities. In conclusion, we present evidence that osteoclasts, both dependent and independent of their resorptive activity, secrete factors stimulating osteoblastic bone formation. © 2012 Elsevier Inc.


PubMed | Uppsala University, Umeå University, Lund Strategic Center for Stem Cell Biology, Regional Cancer Center Uppsala Orebro and Karolinska University Hospital
Type: | Journal: European journal of haematology | Year: 2016

To evaluate the influence of socioeconomic variables on treatment selection and survival of patients with chronic myeloid leukaemia (CML).Using information available in population-based Swedish registries we evaluated indices of health, education and economy from the 980 patients in the Swedish CML register diagnosed between 2002 and 2012. Apart from internal comparisons, five age-, gender- and region-matched control subjects per patient served as control cohort. Median follow-up time from CML diagnosis was 4.8 years.Among CML patients, low personal or household income, short education, living alone, poor performance status and high age (>60yrs) were significantly associated with an inferior survival (in univariate analyses). However, similar findings were noted also in the matched control group, and in comparisons adjusted for calendar year, age and performance status, socioeconomic variables were not significantly associated with CML survival. Meanwhile, both education and income were independently linked to TKI treatment overall and to up-front treatment with second generation TKIs.In conclusion, socioeconomic conditions were associated with survival in the studied CML cohort but these associations could be explained by differences at baseline. Meanwhile, socioeconomic conditions appeared to influence treatment choice. This article is protected by copyright. All rights reserved.


Thudium C.S.,Nordic Bioscience | Moscatelli I.,Lund Strategic Center for Stem Cell Biology | Lofvall H.,Nordic Bioscience | Lofvall H.,Lund Strategic Center for Stem Cell Biology | And 7 more authors.
Calcified Tissue International | Year: 2016

Infantile malignant osteopetrosis (IMO) is a rare, recessive disorder characterized by increased bone mass caused by dysfunctional osteoclasts. The disease is most often caused by mutations in the TCIRG1 gene encoding a subunit of the V-ATPase involved in the osteoclasts capacity to resorb bone. We previously showed that osteoclast function can be restored by lentiviral vector-mediated expression of TCIRG1, but the exact threshold for restoration of resorption as well as the cellular response to vector-mediated TCIRG1 expression is unknown. Here we show that expression of TCIRG1 protein from a bicistronic TCIRG1/GFP lentiviral vector was only observed in mature osteoclasts, and not in their precursors or macrophages, in contrast to GFP expression, which was observed under all conditions. Thus, vector-mediated TCIRG1 expression appears to be post-transcriptionally regulated, preventing overexpression and/or ectopic expression and ensuring protein expression similar to that of wild-type osteoclasts. Codon optimization of TCIRG1 led to increased expression of mRNA but lower levels of protein and functional rescue. When assessing the functional rescue threshold in vitro, addition of 30 % CB CD34+ cells to IMO CD34+ patient cells was sufficient to completely normalize resorptive function after osteoclast differentiation. From both an efficacy and a safety perspective, these findings will clearly be of benefit during further development of gene therapy for osteopetrosis. © 2016 Springer Science+Business Media New York


Moscatelli I.,Lund Strategic Center for Stem Cell Biology | Thudium C.S.,Lund Strategic Center for Stem Cell Biology | Flores C.,Lund Strategic Center for Stem Cell Biology | Schulz A.,University of Ulm | And 10 more authors.
Bone | Year: 2013

Infantile malignant osteopetrosis (IMO) is a rare, lethal, autosomal recessive disorder characterized by non-functional osteoclasts. More than 50% of the patients have mutations in the TCIRG1 gene, encoding for a subunit of the osteoclast proton pump. The aim of this study was to restore the resorptive function of IMO osteoclasts by lentiviral mediated gene transfer of the TCIRG1 cDNA. CD34+ cells from peripheral blood of five IMO patients and from normal cord blood were transduced with lentiviral vectors expressing TCIRG1 and GFP under a SFFV promoter, expanded in culture and differentiated on bone slices to mature osteoclasts. qPCR analysis and western blot revealed increased mRNA and protein levels of TCIRG1, comparable to controls. Vector corrected IMO osteoclasts generated increased release of Ca2+ and bone degradation product CTX-I into the media as well as increased formation of resorption pits in the bone slices, while non-corrected IMO osteoclasts failed to resorb bone. Resorption was approximately 70-80% of that of osteoclasts generated from cord blood. Furthermore, transduced CD34+ cells successfully engrafted in NSG-mice. In conclusion we provide the first evidence of lentiviral-mediated correction of a human genetic disease affecting the osteoclastic lineage. © 2013 Elsevier Inc.

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