Aberrant MNX1 expression associated with t(7;12)(q36;p13) pediatric acute myeloid leukemia induces the disease through altering histone methylation.

Certain subtypes of acute myeloid leukemia (AML) in children have inferior outcome, such as AML with translocation t(7;12)(q36;p13) leading to an MNX1::ETV6 fusion along with high expression of MNX1. We have identified the transforming event in this AML and possible ways of treatment. Retroviral expression of MNX1 was able to induce AML in mice, with similar gene expression and pathway enrichment to t(7;12) AML patient data. Importantly, this leukemia was only induced in immune incompetent mice using fetal but not adult hematopoietic stem and progenitor cells. The restriction in transforming capacity to cells from fetal liver is in alignment with t(7;12)(q36;p13) AML being mostly seen in infants. Expression of MNX1 led to increased histone 3 lysine 4 mono-, di- and trimethylation, reduction in H3K27me3, accompanied with changes in genome-wide chromatin accessibility and genome expression, likely mediated through MNX1 interaction with the methionine cycle and methyltransferases. MNX1 expression increased DNA damage, depletion of the Lin-/Sca1+/c-Kit+ population and skewing toward the myeloid lineage. These effects, together with leukemia development, were prevented by pre-treatment with the S-adenosylmethionine analog Sinefungin. In conclusion, we have shown the importance of MNX1 in development of AML with t(7;12), supporting a rationale for targeting MNX1 and downstream pathways.

Autologous endothelialisation by the stromal vascular fraction on laminin-bioconjugated nanocellulose-alginate scaffolds.

Establishing a vascular network in biofabricated tissue grafts is essential for ensuring graft survival. Such networks are dependent on the ability of the scaffold material to facilitate endothelial cell adhesion; however, the clinical translation potential of tissue-engineered scaffolds is hindered by the lack of available autologous sources of vascular cells. Here, we present a novel approach to achieving autologous endothelialisation in nanocellulose-based scaffolds by using adipose tissue-derived vascular cells on nanocellulose-based scaffolds. We used sodium periodate-mediated bioconjugation to covalently bind laminin to the scaffold surface and isolated the stromal vascular fraction and endothelial progenitor cells (EPCs; CD31+CD45-) from human lipoaspirate. Additionally, we assessed the adhesive capacity of scaffold bioconjugationin vitrousing both adipose tissue-derived cell populations and human umbilical vein endothelial cells. The results showed that the bioconjugated scaffold exhibited remarkably higher cell viability and scaffold surface coverage by adhesion regardless of cell type, whereas control groups comprising cells on non-bioconjugated scaffolds exhibited minimal cell adhesion across all cell types. Furthermore, on culture day 3, EPCs seeded on laminin-bioconjugated scaffolds showed positive immunofluorescence staining for the endothelial markers CD31 and CD34, suggesting that the scaffolds promoted progenitor differentiation into mature endothelial cells. These findings present a possible strategy for generating autologous vasculature and thereby increase the clinical relevance of 3D-bioprinted nanocellulose-based constructs.

An induced pluripotent stem cell t(7;12)(q36;p13) acute myeloid leukemia model shows high expression of MNX1 and a block in differentiation of the erythroid and megakaryocytic lineages.

Acute myeloid leukemia (AML) results from aberrant hematopoietic processes and these changes are frequently initiated by chromosomal translocations. One particular subtype, AML with translocation t(7;12)(q36;p13), is found in children diagnosed before 2 years of age. The mechanisms for leukemogenesis induced by t(7;12) is not understood, in part because of the lack of efficient methods to reconstruct the leukemia-associated genetic aberration with correct genomic architecture and regulatory elements. We therefore created induced pluripotent stem cell (iPSC) lines that carry the translocation t(7;12) using CRISPR/Cas9. These t(7;12) iPSC showed propensity to differentiate into all three germ layers, confirming retained stem cell properties. The potential for differentiation into hematopoietic stem and progenitor cells (HSPC) was shown by expression of CD34, CD43 and CD45. Compared with the parental iPSC line, a significant decrease in cells expressing CD235a and CD41a was seen in the t(7;12) iPSC-derived HSPC (iHSPC), suggesting a block in differentiation. Moreover, colony formation assay showed an accumulation of cells at the erythroid and myeloid progenitor stages. Gene expression analysis revealed significant down-regulation of genes associated with megakaryocyte differentiation and up-regulation of genes associated with myeloid pathways but also genes typically seen in AML cases with t(7;12). Thus, this iPSC t(7;12) leukemia model of the t(7;12) AML subtype constitutes a valuable tool for further studies of the mechanisms for leukemia development and to find new treatment options.

Long-term in vivo survival of 3D-bioprinted human lipoaspirate-derived adipose tissue: proteomic signature and cellular content.

Three-dimensional (3D)-bioprinted lipoaspirate-derived adipose tissue (LAT) is a potential alternative to lipo-injection for correcting soft-tissue defects. This study investigated the long-term in vivo survival of 3D-bioprinted LAT and its proteomic signature and cellular composition. We performed proteomic and multicolour flow cytometric analyses on the lipoaspirate and 3D-bioprinted LAT constructs were transplanted into nude mice, followed by explantation after up to 150 days. LAT contained adipose-tissue-derived stem cells (ASCs), pericytes, endothelial progenitor cells (EPCs) and endothelial cells. Proteomic analysis identified 6,067 proteins, including pericyte markers, adipokines, ASC secretome proteins, proangiogenic proteins and proteins involved in adipocyte differentiation and developmental morphogenic signalling, as well as proteins not previously described in human subcutaneous fat. 3D-bioprinted LAT survived for 150 days in vivo with preservation of the construct shape and size. Furthermore, we identified human blood vessels after 30 and 150 days in vivo, indicating angiogenesis from capillaries. These results showed that LAT has a favourable proteomic signature, contains ASCs, EPCs and blood vessels that survive 3D bioprinting and can potentially facilitate angiogenesis and successful autologous fat grafting in soft-tissue reconstruction.

The aldehyde dehydrogenase cord blood potency assay excludes early apoptotic cells.

BACKGROUND: Cord blood units (CBUs) are processed, frozen, and thawed before use in hematopoietic stem cell (HSC) transplantation. The manipulations affect HSC functionality, that is, induce apoptosis and reduce viability. HSC content, commonly expressed as CBU potency, that is, the expected ability of a CBU to restore hematopoiesis, is traditionally approximated through viable CD34+ cells and the colony-forming unit (CFU) cell cultivation assay. Alternative approaches, for example, the aldehyde dehydrogenase (ALDH) enzyme-based assay, are also forthcoming. We hypothesized that the ALDH assay might exclude apoptotic cells since it is based on enzyme activity. To investigate this, we designed a protocol for simultaneous staining of viable and apoptotic CD34+ and ALDH+ cells using 7-aminoactinomycin (7-AAD) and annexin V, in frozen-thawed CBUs. Results were correlated with results from the colony-forming unit-granulocyte/macrophage (CFU-GM) assay. STUDY DESIGN AND METHODS: Samples from 57 CBUs were thawed and simultaneously analyzed for CD34+ cells, ALDH+ cells, viability (7-AAD), and apoptosis (annexin V) using flow cytometry. Enumeration of CFUs was also performed. RESULTS: No nonviable and few apoptotic cells (mean 0.7%) were identified in the ALDH+ population compared to the viable CD34+ population (mean 3.6%). The total number of ALDH+ cells correlated better than viable CD34+ cells (r = 0. 72 vs. r = 0.66; p < 0.0001) with the results of the CFU assay. CONCLUSION: The ALDH assay excludes nonviable and apoptotic cells, and therefore correlates better with CFU enumeration compared to the number of viable CD34+ cells. We propose that the ALDH assay might replace the CFU-GM method in CBU potency measurements.

Iron sucrose-labeled human mesenchymal stem cells: in vitro multilineage capability and in vivo traceability in a lapine xenotransplantation model.

For evaluation of cell therapy applications, it is of interest to be able to trace and observe cellular distribution of the transplanted cells. The aim with the study was to examine viability, traceability, and multilineage capability of iron sucrose-labeled mesenchymal stem cells (MSCs) after transplantation into lapine intervertebral discs (IVDs). MSCs were collected from three human donors, age 31-50 years, and IVDs from 12 rabbits, age 3 months. MSCs were isolated from the bone marrow and cultured using standard protocols. Iron sucrose labeling of MSCs was performed in Dulbecco's Modified Eagle's Medium-low glucose with Venofer(®). The iron sucrose-labeled MSCs were differentiated into the adipogenic, osteogenic, and chondrogenic lineages. Results were evaluated using Oil red, von Kossa, Alcian blue, and collagen II (immunohistochemistry). For the animal experiments, iron sucrose-labeled MSCs and nonlabeled MSCs were injected into lapine IVDs (LI-LIV level). After transplantation, at the time points of 1 and 3 months, IVDs were collected and cells were analyzed for cell viability (fluorescence-activated cell sorting). The lapine IVDs were collected and examined for presence of cells positive for iron deposits using Berliner blue staining. Differentiation of the iron sucrose-labeled MSCs into adipogenic (lipid droplets), osteogenic (calcium deposits), and chondrogenic lineage (proteoglycan/collagen II accumulation) (3/3 donors) was observed in vitro. After transplantation, the mean cell viability for iron-labeled MSCs/IVD cells was 99%, for nonlabeled MSCs/IVD cells was 95%, and for control IVD cells was 99% at a time point of 1 month. At a time point of 3 months, mean cell viability was 73% for iron sucrose-labeled MSCs/IVD cells, for nonlabeled MSCs/IVD cells was 77%, and for control IVD cells was 98%. At the time point of 1 month, cells positive for iron deposits were detected sparsely distributed in IVDs (tissue sections) in 4/4 animals and at the time point of 3 months in 4/4 animals. The results indicate that iron sucrose can be used as a cell tracer with a stable detection potential in tissues (histologies). This may be an important evaluation tool for understanding stem cell distribution/function after transplantation into degenerated cartilaginous tissues.

Exploring the heterogeneity of the hematopoietic stem and progenitor cell pool in cord blood: simultaneous staining for side population, aldehyde dehydrogenase activity, and CD34 expression.

BACKGROUND: The stem cell content in cord blood (CB) units is routinely assessed regarding nucleated cells, CD34+ cell count, and number of colony-forming units (CFUs). Efforts are made toward finding better ways of defining stemness of CB units. Side population (SP) phenotype and activity of aldehyde dehydrogenase (ALDH) are functional markers of stemness that can be assayed using flow cytometry. STUDY DESIGN AND METHODS: We have developed a protocol for simultaneous determination of CD34+, SP, and ALDH+ populations in relation to immature white blood cells (CD45dim) in CB. Viable nucleated cells were consecutively stained for SP and ALDH activity and with antibodies against the CD45, CD34, and CD117 antigens. RESULTS: The SP and ALDH+ populations could reliably be measured simultaneously. The median sizes of the SP and the ALDH+ populations were 0.85 and 3.3% of CD45dim cells, respectively. There was no overlap between the SP and ALDH+ populations. Cells that were ALDH+ expressed CD34 and CD117, but SP cells were negative for these markers. The ALDH+ cell content correlated with CD34+ cell content (p < 0.001) and with CFU-granulocyte-macrophage (GM; p = 0.03) but not with total CFUs. SP did not correlate with CD34+, CFU-GM, or total CFU. CONCLUSIONS: We show that simultaneous detection of the CD34, SP, and ALDH+ cells is clearly feasible using only small amounts of CB. In CB, ALDH+, and CD34+ cells are overlapping populations distinctly separated from the SP population. The difference in relation to the capacity for colony growth between ALDH+ and SP underlines that they define different cell populations.

p27(KIP1) and PTEN cooperate in myeloproliferative neoplasm tumor suppression in mice.

PTEN acts as a phosphatase for PIP3 and negatively regulates the PI3K/AKT pathway, and p27(KIP1) is a cyclin-dependent kinase inhibitor that regulates the G1 to S-phase transition by binding to and regulating the activity of cyclin-dependent kinases. Genetic alterations of PTEN or CDKN1B (p27(KIP1)) are common in hematological malignancies. To better understand how mutations in these two genes might cooperate in leukemogenesis, we inactivated both genes in the hematological compartment in mice. Here, we show that the combined inactivation of Pten and Cdkn1b results in a more severe myeloproliferative neoplasm phenotype associated with lower hemoglobin, enlarged spleen and liver, and shorter lifespan compared to inactivation of Pten alone. More severe anemia and increased myeloid infiltration and destruction of the spleen contributed to the earlier death of these mice, and elevated p-AKT, cyclin D1, and cyclin D3 might contribute to the development of this phenotype. In conclusion, PTEN and p27(KIP1) cooperate in tumor suppression in the hematological compartment.

Proteomic profiling of cerebrospinal fluid in parkinsonian disorders.

Parkinson's disease (PD) and atypical parkinsonian disorders (APD), including multiple system atrophy (MSA), progressive supranuclear palsy (PSP), and corticobasal degeneration (CBD), are a group of neurodegenerative diseases sharing many similar signs and symptoms but distinguished by their particular clinical features, treatment response, prognosis and mortality. The differential diagnosis may be challenging, especially in early disease stages. Considering the importance of an accurate diagnosis both for clinical management and for research, new diagnostic tools are needed. In this study, we investigated 56 PD, 42 MSA, 39 PSP, 9 CBD patients, and 24 healthy controls. After screening the cerebrospinal fluid (CSF) proteome using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS), we identified 4 proteins (ubiquitin [mass-to-charge ratio (m/z) 8590], beta2-microglobulin [m/z 11730], and 2 secretogranin 1 [chromogranin B] fragments [m/z 7260 and m/z 6250]) that differentiated healthy controls and PD patients from patients with APD. However, they could not differentiate PD patients from controls. As none of these changes were APD subgroup-specific, they most likely reflect the intensity and/or extent of the neurodegenerative process in general.

Novel cerebrospinal fluid biomarkers of axonal degeneration in frontotemporal dementia.

Frontotemporal dementia (FTD) is a heterogeneous disease with substantial interpersonal variance in aggressiveness. Novel biomarkers for rapidly progressive FTD could improve diagnosis and provide clues regarding its pathogenesis. In this study, surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS) was used to analyze peptide profiles in cerebrospinal fluid (CSF) from 24 FTD patients. Thirteen patients had rapidly progressive FTD with distinct pathology in a brain MRI after less than 3 years of disease duration. Eleven patients had slowly progressive FTD with a normal brain MRI, but had abnormal findings in SPECT/PET after more than 5 years of disease duration. The axonal damage marker CSF neurofilament light-chain (NF-L) was measured in all subjects to evaluate the amount of axonal degeneration. A CSF NF-L level of 150 ng/l was used as a cut-off point for high NF-L expression. SELDI-TOF analysis of peptides in the range of 2000-20000 m/z revealed one peak with m/z of 6378 that was expressed at a significantly different level (p<0.01) when rapidly versus slowly progressive cases of FTD were compared. Eleven peaks were expressed at different levels when high versus low CSF NF-L were compared. Using chromatographic purification followed by tandem mass spectrometric analysis, five of these peaks were identified as follows: C-terminal fragment of neuroendocrine protein 7B2 (3512.84 Da), C-terminal fragment of osteopontin (7658.19 Da) as well as its mono- and diphosphorylated forms (7738.16 Da and 7818.13 Da, respectively) and pancreatic ribonuclease (14566.33 Da). The peak intensity of pancreatic ribonuclease was higher in patients with low NF-L expression, while the other peptides had a lower peak intensity in this group. Altered levels of these peptides have also been described in other neurodegenerative diseases. Taken together, these data suggest that differentially-expressed peptides are general markers of axonal degeneration. Further studies are needed to verify their prognostic value in FTD.

Cerebrospinal fluid concentrations of peptides derived from chromogranin B and secretogranin II are decreased in multiple sclerosis.

Novel biomarkers for multiple sclerosis (MS) could improve diagnosis and provide clues to pathogenesis. In this study surface-enhanced laser desorption/ionization time-of-flight mass spectrometry was used to analyze protein expression in CSF from 46 MS patients, 46 healthy siblings to the patients, and 50 unrelated healthy controls. Twenty-four proteins in the mass range 2-10 kDa were expressed at significantly different levels (p < 0.01) in a robust manner when comparing the three groups. Identities of three proteins were determined using biochemical purification followed by tandem mass spectrometric analysis. Immunoprecipitation experiments confirmed the identities for two peptides derived from chromogranin B (m/z 6252) and from secretogranin II (m/z 3679). These peptides were all decreased in MS when compared with siblings or controls. Radioimmunoassays specific for each peptide confirmed these differences. The lowered concentrations did not correlate to the axonal damage marker neurofilament light protein and may thus reflect functional changes rather than neurodegeneration. Further studies will investigate the involvement of these peptides in MS pathogenesis.

Activity of the LMP1 gene promoter in Epstein-Barr virus-transformed cell lines is modulated by sequence variations in the promoter-proximal CRE site.

The Epstein-Barr virus (EBV)-encoded tumour-associated latent membrane protein 1 (LMP1) gene expression is transactivated by EBV nuclear antigen 2 (EBNA2) in human B cells. We have previously identified a cyclic AMP-responsive element (CRE) in the B95-8 LMP1 promoter that is essential for transcription activation. Sequencing of LMP1 promoter in the P3HR1-derived EREB2.5 cell line revealed 25 single base pair substitutions in comparison to the B95-8 virus, one of them localized in the CRE element. Sequence variations in this element have been identified in several EBV isolates of both African and Asian origins. The effect of the P3HR1 CRE site variation on binding of factors to the LMP1 promoter sequence (LRS) and promoter activation was investigated with electrophoretic mobility-shift assays and reporter gene transfection assays. ATF1 and CREB1 transcription factors bound with reduced efficiency to the P3HR1 variant and below the detection level to the other tested variants. Accordingly, reporter plasmids carrying the P3HR1 CRE sequence in a B95-8 LRS context displayed 50 % lower activity in all tested cell lines. The impaired ability to activate transcription caused by the C to A substitution in CRE was not apparent when the mutated site was placed in a P3HR1 LRS context and the reporter transfected into Jijoye cells, most likely as a consequence of the other base pair substitutions in P3HR1 LRS. Overall, our results suggest that the mutations in the LRS CRE site have been conserved to adjust LMP1 expression to levels that favour cell survival in certain cellular and environmental contexts.

Identification of CSF biomarkers for frontotemporal dementia using SELDI-TOF.

This investigation describes the discovery of novel possible cerebrospinal fluid (CSF) biomarkers for frontotemporal dementia (FTD) using surface-enhanced laser desorption/ionization time-of-flight (SELDI-TOF) mass spectrometry (MS). Sixteen clinically diagnosed FTD patients and 12 non-demented controls were included in the study. CSF was collected and analyzed for protein expression by SELDI-TOF MS. The samples were analyzed on four different array surfaces using two different energy-absorbing molecules as matrices. In total each sample was subjected to eight different surface/matrix conditions. About 2000 protein peaks (mass/charge ratios) were detected. Forty-two peaks were differentially expressed in FTD (P < 0.01). After exclusion of peaks with low signal-to-noise ratio and/or poor resolution and peaks representing differentially charged proteins, 10 peaks remained, five of which were increased and five decreased in FTD cases compared to controls. Using partial least square discriminant analysis (PLS-DA), the combination of these biomarkers discriminated FTD from non-demented controls with a sensitivity of 94%, a specificity of 83% and an accuracy of 89%. Five of the peaks were purified further and identified by tandem MS as a fragment of neurosecretory protein VGF, transthyretin, S-cysteinylated transthyretin, truncated cystatin C and a fragment of chromogranin B. With use of these potential biomarkers, FTD can be distinguished from control subjects with high accuracy in this pilot study.