Ola Hermanson

Tracheobronchial transplantation with a stem-cell-seeded bioartificial nanocomposite: a proof-of-concept study

BACKGROUND Tracheal tumours can be surgically resected but most are an inoperable size at the time of diagnosis; therefore, new therapeutic options are needed. We report the clinical transplantation of the tracheobronchial airway with a stem-cell-seeded bioartificial nanocomposite. METHODS A 36-year-old male patient, previously treated with debulking surgery and radiation therapy, presented with recurrent primary cancer of the distal trachea and main bronchi. After complete tumour resection, the airway was replaced with a tailored bioartificial nanocomposite previously seeded with autologous bone-marrow mononuclear cells via a bioreactor for 36 h. Postoperative granulocyte colony-stimulating factor filgrastim (10 μg/kg) and epoetin beta (40,000 UI) were given over 14 days. We undertook flow cytometry, scanning electron microscopy, confocal microscopy epigenetics, multiplex, miRNA, and gene expression analyses. FINDINGS We noted an extracellular matrix-like coating and proliferating cells including a CD105+ subpopulation in the scaffold after the reseeding and bioreactor process. There were no major complications, and the patient was asymptomatic and tumour free 5 months after transplantation. The bioartificial nanocomposite has patent anastomoses, lined with a vascularised neomucosa, and was partly covered by nearly healthy epithelium. Postoperatively, we detected a mobilisation of peripheral cells displaying increased mesenchymal stromal cell phenotype, and upregulation of epoetin receptors, antiapoptotic genes, and miR-34 and miR-449 biomarkers. These findings, together with increased levels of regenerative-associated plasma factors, strongly suggest stem-cell homing and cell-mediated wound repair, extracellular matrix remodelling, and neovascularisation of the graft. INTERPRETATION Tailor-made bioartificial scaffolds can be used to replace complex airway defects. The bioreactor reseeding process and pharmacological-induced site-specific and graft-specific regeneration and tissue protection are key factors for successful clinical outcome. FUNDING European Commission, Knut and Alice Wallenberg Foundation, Swedish Research Council, StratRegen, Vinnova Foundation, Radiumhemmet, Clinigene EU Network of Excellence, Swedish Cancer Society, Centre for Biosciences (The Live Cell imaging Unit), and UCL Business.

High susceptibility of neural stem cells to methylmercury toxicity: effects on cell survival and neuronal differentiation

Neural stem cells (NSCs) play an essential role in both the developing embryonic nervous system through to adulthood where the capacity for self‐renewal may be important for normal function of the CNS, such as in learning, memory and response to injury. There has been much excitement about the possibility of transplantation of NSCs to replace damaged or lost neurones, or by recruitment of endogenous precursors. However, before the full potential of NSCs can be realized, it is essential to understand the physiological pathways that control their proliferation and differentiation, as well as the influence of extrinsic factors on these processes. In the present study we used the NSC line C17.2 and primary embryonic cortical NSCs (cNSCs) to investigate the effects of the environmental contaminant methylmercury (MeHg) on survival and differentiation of NSCs. The results show that NSCs, in particular cNSCs, are highly sensitive to MeHg. MeHg induced apoptosis in both models via Bax activation, cytochrome c translocation, and caspase and calpain activation. Remarkably, exposure to MeHg at concentrations comparable to the current developmental exposure (via cord blood) of the general population in many countries inhibited spontaneous neuronal differentiation of NSCs. Our studies also identified the intracellular pathway leading to MeHg‐induced apoptosis, and indicate that NSCs are more sensitive than differentiated neurones or glia to MeHg‐induced cytotoxicity. The observed effects of MeHg on NSC differentiation offer new perspectives for evaluating the biological significance of MeHg exposure at low levels.

Estrogen-induced alterations of spinal cord enkephalin gene expression.

Enkephalin-synthesizing neurons in the superficial laminae of the spinal and trigeminal dorsal horn are critical components of the endogenous pain-modulatory system. We have previously demonstrated that these neurons display intracellular estrogen receptors, suggesting that estrogen can potentially influence their enkephalin expression. By using Northern blot, we now show that a bolus injection of estrogen results in a rapid increase in spinal cord enkephalin mRNA levels in ovariectomized female rats. Thus, 4 h after estrogen administration the enkephalin mRNA-expression in the lumbar spinal cord was on average 68% higher (P<0.05) than in control animals injected with vehicle only. A small increase in the amount of enkephalin mRNA was also seen after 8 h (P<0.05), whereas no difference between estrogen-injected and control animals was found after 24 h or at time periods shorter than 4 h. Taken together with the previous anatomical data, the present findings imply that estrogen has an acute effect on spinal opioid levels in areas involved in the transmission of nociceptive information.

The histone H4 lysine 16 acetyltransferase hMOF regulates the outcome of autophagy

Autophagy is an evolutionarily conserved catabolic process involved in several physiological and pathological processes. Although primarily cytoprotective, autophagy can also contribute to cell death; it is thus important to understand what distinguishes the life or death decision in autophagic cells. Here we report that induction of autophagy is coupled to reduction of histone H4 lysine 16 acetylation (H4K16ac) through downregulation of the histone acetyltransferase hMOF (also called KAT8 or MYST1), and demonstrate that this histone modification regulates the outcome of autophagy. At a genome-wide level, we find that H4K16 deacetylation is associated predominantly with the downregulation of autophagy-related genes. Antagonizing H4K16ac downregulation upon autophagy induction results in the promotion of cell death. Our findings establish that alteration in a specific histone post-translational modification during autophagy affects the transcriptional regulation of autophagy-related genes and initiates a regulatory feedback loop, which serves as a key determinant of survival versus death responses upon autophagy induction.

Estrogen receptor-like immunoreactivity in the medullary and spinal dorsal horn of the female rat

Using an immunohistochemical technique, we demonstrate that large numbers of neurons in the laminar spinal trigeminal nucleus and spinal gray matter of the female rat express estrogen receptors (ER). Densely packed ER-immunoreactive neurons were present in lamina II, but labeled neurons were also present in lamina I, the neck of the dorsal horn, and in lamina X. Labeling was present throughout the length of the spinal cord, with the exception of segments caudal to S1, which were unlabeled. The distribution of ER-containing neurons to areas that are involved in processing of primary afferent nociceptive information suggests that the pain modulatory effects of estrogen may be exerted at the spinal level.

Colocalization of oestrogen receptor immunoreactivity and preproenkephalin mRNA expression to neurons in the superficial laminae of the spinal and medullary dorsal horn of rats.

A double‐labelling procedure combining immunohistochemical staining with in situ hybridization using a radiolabelled cRNA probe was employed to demonstrate oestrogen receptor‐like immunoreactivity and preproenkephalin‐A mRNA in the medullary and spinal dorsal horn of female rats. Both markers labelled large numbers of neurons in the substantia gelatinosa and its trigeminal homologue. Many of these neurons were double‐labelled, displaying both oestrogen receptor‐like‐ immunoreactivity and preproenkephalin‐A mRNA; cell counts showed that 40–60% of the of the oestrogen receptor‐like‐immunoreactive cells in the superficial laminae also were labelled for preproenkephalin‐A mRNA, and that 60–70% of the preproenkephalin‐A mRNA‐labelled neurons in the same laminae displayed oestrogen receptor‐like immunoreactivity. Previous studies have shown that oestrogen receptors can bind to the promoter region of the preproenkephalin‐A gene, and studies on the hypothalamus have demonstrated that oestrogen regulates enkephalin expression in select neuronal populations. The present results demonstrate that enkephalinergic neurons in the superficial dorsal horn contain oestrogen receptors and suggest that oestrogen may play an important role in the modulation of sensory and nociceptive processing in the lower medulla and spinal cord.

Subnuclear localization of FOS‐like immunoreactivity in the rat parabrachial nucleus after nociceptive stimulation

The effect of noxious stimulation on the expression of FOS‐like immunoreactivity (FOS‐LI) in neurons of the parabrachial nucleus (PB) was studied in awake, freely moving rats. In one series of experiments, the rats were subjected to noxious mechanical stimulation (pinch) of either the nape of the neck or the base of the tail for 20 seconds every 5 minutes for 90 minutes, and then they were killed by transcardial perfusion after 45–210 minutes. Control animals received innocuous mechanical stimulation (brush) of the tail. Noxious stimuli resulted in FOS‐LI in neurons in the dorsal part of the lateral PB, with heavy labeling in the superior lateral (PBsl) and the dorsal lateral (PBdl) subnuclei. FOS‐LI was also elicited in the central lateral subnucleus (PBcl) and, although much more sparsely, in the external lateral subnucleus and the Kölliker‐Fuse nucleus. Tail and neck stimulation resulted in similar labeling patterns, but more neurons, particularly in PBsl, expressed FOS‐LI after pinch of the tail than of the neck.

CXXC5 Is a Novel BMP4-regulated Modulator of Wnt Signaling in Neural Stem Cells

Bone morphogenetic proteins such as BMP4 are essential for proper development of telencephalic forebrain structures and induce differentiation of telencephalic neural stem cells into a variety of cellular fates, including astrocytic, neuronal, and mesenchymal cells. Little is yet understood regarding the mechanisms that underlie the spatiotemporal differences in progenitor response to BMP4. In a screen designed to identify novel targets of BMP4 signaling in telencephalic neural stem cells, we found the mRNA levels of the previously uncharacterized factor CXXC5 reproducibly up-regulated upon BMP4 stimulation. In vivo, CXXC5 expression overlapped with BMP4 adjacent to Wnt3a expression in the dorsal regions of the telencephalon, including the developing choroid plexus. CXXC5 showed partial homology with Idax, a related protein previously shown to interact with the Wnt-signaling intermediate Dishevelled (Dvl). Indeed CXXC5 and Dvl co-localized in the cytoplasm and interacted in co-immunoprecipitation experiments. Moreover, fluorescence resonance energy transfer (FRET) experiments verified that CXXC5 and Dvl2 were located in close spatial proximity in neural stem cells. Studies of the functional role of CXXC5 revealed that overexpression of CXXC5 or exposure to BMP4 repressed the levels of the canonical Wnt signaling target Axin2, and CXXC5 attenuated Wnt3a-mediated increase in TOPflash reporter activity. Accordingly, RNA interference of CXXC5 attenuated the BMP4-mediated decrease in Axin2 levels and facilitated the response to Wnt3a in neural stem cells. We propose that CXXC5 is acting as a BMP4–induced inhibitor of Wnt signaling in neural stem cells.

Getting the right stuff: controlling neural stem cell state and fate in vivo and in vitro with biomaterials.

Stem cell therapy holds great promises in medical treatment by, e.g., replacing lost cells, re-constitute healthy cell populations and also in the use of stem cells as vehicles for factor and gene delivery. Embryonic stem cells have rightfully attracted a large interest due to their proven capacity of differentiating into any cell type in the embryo in vivo. Tissue-specific stem cells are however already in use in medical practice, and recently the first systematic medical trials involving human neural stem cell (NSC) therapy have been launched. There are yet many obstacles to overcome and procedures to improve. To ensure progress in the medical use of stem cells increased basic knowledge of the molecular mechanisms that govern stem cell characteristics is necessary. Here we provide a review of the literature on NSCs in various aspects of cell therapy, with the main focus on the potential of using biomaterials to control NSC characteristics, differentiation, and delivery. We summarize results from studies on the characteristics of endogenous and transplanted NSCs in rodent models of neurological and cancer diseases, and highlight recent advancements in polymer compatibility and applicability in regulating NSC state and fate. We suggest that the development of specially designed polymers, such as hydrogels, is a crucial issue to improve the outcome of stem cell therapy in the central nervous system.

Subnuclear localization of FOS‐like immunoreactivity in the parabrachial nucleus after orofacial nociceptive stimulation of the awake rat

We used FOS‐like immunohistochemistry to detect neuronal activity in the pontine parabrachial nucleus after injection of formalin into the lower lip of the awake rat and compared the labeling pattern with that seen after formalin injection into the hindpaw. One hour after a formalin injection into the lip, many FOS‐immunoreactive cells were seen in the parabrachial nucleus, preferentially on the side ipsilateral to the injection site. Detailed anatomical analysis revealed that FOS‐immunoreactive neurons were localized predominantly to three regions of the parabrachial nucleus: the external lateral, the external medial, and the Kölliker‐Fuse subnuclei, with sparser labeling present in the dorsal and superior lateral subnuclei and in the medial parabrachial nucleus. In contrast, a formalin injection into the hindpaw resulted in dense FOS‐labeling in the superior, dorsal, and central lateral subnuclei, with sparse to moderate labeling in the Kölliker‐Fuse nucleus, and sparse labeling in the external lateral and external medial subnuclei, as described previously (Hermanson and Blomqvist, J. Comp. Neurol., [1996] 368:45–56).