Human Hair Follicles: “Bulging” with Neural Crest–Like Stem Cells

Several studies have reported the existence of precursor cells residing within various adult tissues that appear to either retain or recapitulate features of neural crest stem cells (NCSCs). In rodents, unique populations of both epidermal and dermal cells, resident within hair follicles, exhibit such characteristics, although the existence of equivalent NCSC-like cells in human tissues has remained uncertain. In this issue, Yu et al. show that NCSC-like cells also reside within the bulge region of adult human hair follicles

Defining The Role of Primary Cilia on Skin Derived Precursors.

Isolated in 2001, Skin Derived Precursors (SKPs) represent a novel population of multipotent stem cells1,2 residing at the base of hair follicle where they play a key role in defining the physiology and regeneration capacity of hair follicles and skin (Biernaskie et al. 2009). In order to understand how SKPs behavior is regulated in the skin, we asked whether primary cilia, microtubule bundles projected from the cell surface that transmit chemical signals between cells4, are present in the dermal papilla and dermal sheath in-vivo and in the population of isolated SKPs. In our investigation, we performed immunohistochemistry for acetylated tubulin and showed that SKPs do have cilia. We then tested whether these cilia can be elongated following treatments of lithium chloride on these dermal precursors. We then asked whether elongation of cilia has an effect on the self-renewal capacity of SKPs and whether drugs elongating cilia can work synergistically with Platelet Derived Growth Factor (PDGF), a growth factor that we have previously shown to improve cell growth and cell division. Experiments assessing the self-renewal capacity of SKPs suggested significant  increase in the diameter and the number of spherical presence when treated with lithium chloride and when lithium chloride is added in combination with PDGFβ when compared to PDGFβ alone. This suggests that signaling in cilia may influence PDGF signals causing an enhanced effect on SKP proliferation. Further experiments including knocking down primary cilia by blocking the transcription of ciliary protein using shRNA and in-vivo transplantations of lithium chloride and PDGF treated SKPs in a hair follicle formation assays will be executed to understand the key roles of primary cilia on SKPs. These studies will ultimately aim to answer whether drugs affecting cilia can function as potential therapeutic targets for autologous adult stem cell based therapies

METABOLISM OF SKIN DERIVED PRECURSOR (SKP) CELLS IN STIRRED SUSPENSION BIOREACTORS

INTRODUCTION More than six million people suffer from burn injuries every year. These injuries can result in psychological trauma, disabilities and permanent disfigurement. A common treatment for burn victims is an autologous graft surgery in which skin is transplanted from a healthy part of the body to the injury site (i.e. split thickness skin graft). This graft, however, does not contain functional dermal tissue, hair follicles or glands, often causing graft contraction, chronic irritation, and unnatural in appearance. We have hypothesized that skin-derived precursor (SKP) cells, a multipotent dermal stem cell that resides within skin hair follicles, can be utilized in conjunction with split thickness skin grafts to improve their function and minimize irritation. SKPs have a high proliferative potential and need to be expanded in a well-controlled, standardized culture environment before they can be utilized in clinical treatments [1]. It is essential to optimize the expansion of SKP cells in order to generate a bioprocess capable of producing enough cells for a clinical setting. METHODS -qNUTR = ∆[NUTR]/Int(Xv)dtThe specific uptake and production rates were calculated for cells cultured in static T-Flask environments and stirred suspension bioreactors run at 40, 60, 80, and 100 rpm. Equation 1 was used to calculate specific rates (qNUTR). The integral change in viable cells (Int(Xv)dt) was calculated using a numerical trapezoid approximation, and the change in nutrient concentration (∆[NUTR]) was measured using the Nova Bioprofile 100+ analyzer. SKP cells were taken from a 68 year old female. RESULTS The specific uptake rates of glucose and glutamine and specific production rates of lactate and ammonia have been determined under altered cell culture environments for SKP cells (Figure1). This provides details into nutrient limitations and cell metabolic behaviours needed to access parameters to guide our bioprocess design and development of robust expansion protocols. DISCUSSION AND CONCLUSIONS Low levels of oxygen and nutrients result in significant changes to cell growth rates [2].  We were able to conclude that SKP cell growth is not limited by the glucose or glutamine concentrations in the media, and lactate and ammonia do not reach toxic levels. It is interesting to note that the stirred suspension environment does appear to have an effect on the specific consumption rates of glucose and glutamine. According to these initial results, we are predicting that under shear stress environments, SKP cells are changing their metabolic behaviour to allow more glucose to convert to pyruvate and enter the TCA cycle. The specific production rates of lactate and ammonia, however, do not follow the same patterns. Further validation and reasons behind these differences need to be investigated

Development of an alternative harvesting method using pH to detach adherent cells from microcarriers

Peripheral nerve injuries are common in Canada, affecting 2.8% of trauma patients treated every year. Current repair strategies are inadequate and repair is often suboptimal with only 25% of patients recovering full motor function and only 3% regaining full sensory function. Because of this, the field is turning toward regenerative medicine to develop a cellular therapy using Schwann cells to repair injured nerves. Schwann cells differentiated from skin derived precursors (SKP-SCs) are a promising cell type as they are easily obtained and allow for autologous therapy. To be able to generate clinically relevant numbers of SKP-SCs, bioreactors need to be used. Since SKP-SCs are an adherent cell type, to be expanded in suspension bioreactors, small spherical beads known as microcarriers need to be used. Our lab has previously shown that these SKP-SCs readily attach to the microcarriers and grow in stirred suspension bioreactors. We have also shown that by controlling the culture parameters, we can increase the maximum cell density compared to conventional static culture methods. One of the biggest hurdles that remains is an efficient harvesting method that can be scaled up to clinical applications. Current cell detachment protocols use enzymatic based solutions to remove the cells from the surface of the microcarriers. These methods work well in removing the cells, however, they are very labour intensive as they require many washing steps and taking the reactors offline. Therefore, we looked into an alternative method for the detachment of SKP-SCs from microcarriers that will allow for an inline detachment process. This new method is based on previous research done in our lab using high pH solutions to dissociate aggregates. First we investigated the detachment efficiency in static. Cells were cultured in 6-well plates until confluency and then harvested with solutions ranging from pH 8-9.5. With a pH of 9 and an incubation time of 30 minutes, we were able to recover 75% of cells when compared to traditional enzymatic harvesting. Following this we performed a qualitative analysis on the detachment of the SKP-SCs from the microcarriers to determine if this method has potential. Small 3mL samples were taken and solutions with pHs 8.5, 9, and 9.5 were added and incubated for 30 minutes and agitated every 5 minutes. We found that the cells detached with a high efficiency after 30 minutes with a pH of only 8.5. This was then quantified while maintaining a viability of above 90%. Following this we tested this method in harvesting full 125mL bioreactors. We evaluated different pH, agitation rates, and incubation times. We also assessed the ability of the cells to reattach to microcarriers and continue to expand over several serial passages to ensure there were no negative effects on the cells. Lastly we looked at using this method in our controlled bioreactors to increase the pH without the addition of anything else. Based on our results, increasing the pH of the culture medium can detach the SKP-SCs from microcarriers at a pH as low as 8.5 which allows for minimal cell damage while still detaching cells. We also noted that when the pH gets too high (\u3e9.5), the microcarriers begin to clump together causing large aggregates of microcarriers which could lead to clogging during the filtration steps. With increasing agitation, higher recovery efficiencies can be achieved indicating that this method of cell detachment has potential for large volume processes

Factors Within the Endoneurial Microenvironment Act to Suppress Tumorigenesis of MPNST

Background: Deciphering avenues to adequately control malignancies in the peripheral nerve will reduce the need for current, largely-ineffective, standards of care which includes the use of invasive, nerve-damaging, resection surgery. By avoiding the need for en bloc resection surgery, the likelihood of retained function or efficient nerve regeneration following the control of tumor growth is greater, which has several implications for long-term health and well-being of cancer survivors. Nerve tumors can arise as malignant peripheral nerve sheath tumors (MPNST) that result in a highly-aggressive form of soft tissue sarcoma. Although the precise cause of MPNST remains unknown, studies suggest that dysregulation of Schwann cells, mediated by the microenvironment, plays a key role in tumor progression. This study aimed to further characterize the role of local microenvironment on tumor progression, with an emphasis on identifying factors within tumor suppressive environments that have potential for therapeutic application.Methods: We created GFP-tagged adult induced tumorigenic Schwann cell lines (iSCs) and transplanted them into various in vivo microenvironments. We used immunohistochemistry to document the response of iSCs and performed proteomics analysis to identify local factors that might modulate divergent iSC behaviors.Results: Following transplant into the skin, spinal cord or epineurial compartment of the nerve, iSCs formed tumors closely resembling MPNST. In contrast, transplantation into the endoneurial compartment of the nerve significantly suppressed iSC proliferation. Proteomics analysis revealed a battery of factors enriched within the endoneurial compartment, of which one growth factor of interest, ciliary neurotrophic factor (CNTF) was capable of preventing iSCs proliferation in vitro.Conclusions: This dataset describes a novel approach for identifying biologically relevant therapeutic targets, such as CNTF, and highlights the complex relationship that tumor cells have with their local microenvironment. This study has significant implications for the development of future therapeutic strategies to fight MPNSTs, and, consequently, improve peripheral nerve regeneration and nerve function

Cystatin C is glucocorticoid responsive, directs recruitment of Trem2+ macrophages, and predicts failure of cancer immunotherapy

Cystatin C (CyC), a secreted cysteine protease inhibitor, has unclear biological functions. Many patients exhibit elevated plasma CyC levels, particularly during glucocorticoid (GC) treatment. This study links GCs with CyC’s systemic regulation by utilizing genome-wide association and structural equation modeling to determine CyC production genetics in the UK Biobank. Both CyC production and a polygenic score (PGS) capturing predisposition to CyC production were associated with increased all-cause and cancer-specific mortality. We found that the GC receptor directly targets CyC, leading to GC-responsive CyC secretion in macrophages and cancer cells. CyC-knockout tumors displayed significantly reduced growth and diminished recruitment of TREM2+ macrophages, which have been connected to cancer immunotherapy failure. Furthermore, the CyC-production PGS predicted checkpoint immunotherapy failure in 685 patients with metastatic cancer from combined clinical trial cohorts. In conclusion, CyC may act as a GC effector pathway via TREM2+ macrophage recruitment and may be a potential target for combination cancer immunotherapy.publishedVersio

macroH2A2 antagonizes epigenetic programs of stemness in glioblastoma

Self-renewal is a crucial property of glioblastoma cells that is enabled by the choreographed functions of chromatin regulators and transcription factors. Identifying targetable epigenetic mechanisms of self-renewal could therefore represent an important step toward developing effective treatments for this universally lethal cancer. Here we uncover an epigenetic axis of self-renewal mediated by the histone variant macroH2A2. With omics and functional assays deploying patient-derived in vitro and in vivo models, we show that macroH2A2 shapes chromatin accessibility at enhancer elements to antagonize transcriptional programs of self-renewal. macroH2A2 also sensitizes cells to small molecule-mediated cell death via activation of a viral mimicry response. Consistent with these results, our analyses of clinical cohorts indicate that high transcriptional levels of this histone variant are associated with better prognosis of high-grade glioma patients. Our results reveal a targetable epigenetic mechanism of self-renewal controlled by macroH2A2 and suggest additional treatment approaches for glioblastoma patients

Enriched Rehabilitative Training Promotes Improved Forelimb Motor Function and Enhanced Dendritic Growth after Focal Ischemic Injury

Chronic impairment of forelimb and digit movement is a common problem after stroke that is resistant to therapy. Previous studies have demonstrated that enrichment improves behavioral outcome after focal ischemia; however, postischemic enrichment alone is not capable of enhancing fine digit and forelimb function. Therefore, we combined environmental enrichment with daily skilled-reach training to assess the effect of intensive task-specific rehabilitation on long-term functional outcome. Rats were subjected to either endothelin-1-induced focal ischemia or sham surgery and subsequently designated to enriched-rehabilitation or standard-housing treatment groups starting 15 d after ischemia. Functional assessment of the affected forelimb at 4 and 9 weeks after treatment revealed that ischemic plus enrichment (IE) animals had improved ∼30% on the staircase-reaching task and were indistinguishable from sham animals for both latency and foot faults in a beam-traversing task. In contrast, ischemic plus standard (IS) animals remained significantly impaired on both tasks. Interestingly, both ischemic groups (IE and IS) relied on the nonaffected forelimb during upright weight-bearing movements, a pattern that persisted for the duration of the experiment. Dendritic arborization of layer V pyramidal cells within the undamaged motor cortex was examined using a Golgi-Cox procedure. IE animals showed enhanced dendritic complexity and length compared with both IS and sham groups. These results suggest that enrichment combined with task-specific rehabilitative therapy is capable of augmenting intrinsic neuronal plasticity within noninjured, functionally connected brain regions, as well as promoting enhanced functional outcome

Efficacy of Rehabilitative Experience Declines with Time after Focal Ischemic Brain Injury

To maximize the effectiveness of rehabilitative therapies after stroke, it is critical to determine when the brain is most responsive (i.e., plastic) to sensorimotor experience after injury and to focus such efforts within this period. Here, we compared the efficacy of 5 weeks of enriched rehabilitation (ER) initiated at 5 d (ER5), ER14, or ER30 after focal ischemia, as judged by functional outcome and neuromorphological change. ER5 provided marked improvement in skilled forelimb reaching ability and ladder-rung- and narrow-beam-walking tasks and attenuated the stroke-induced reliance on the unaffected forepaw for postural support. ER14 provided improvement to a somewhat lesser extent, whereas recovery was diminished after ER30 such that motor function did not differ from ischemic animals exposed to social housing. To examine potential neural substrates of the improved function, we examined dendritic morphology in the undamaged motor cortex because our previous work (Biernaskie and Corbett, 2001) suggested that recovery was associated with enhanced dendritic growth in this region. ER5 increased the number of branches and complexity of layer V neurons compared with both social housing and control animals. Dendritic arbor after ER14 (although increased) and ER30 did not differ from those exposed to social housing. These data suggest that the poststroke brain displays heightened sensitivity to rehabilitative experience early after the stroke but declines with time. These findings have important implications for rehabilitation of stroke patients, many of whom experience considerable delays before therapy is initiated