Choroid plexus (CP) supports the entry of monocyte-derived macrophages (MDMs) to the central nervous system in animal models of traumatic brain injury, spinal cord injury, and Alzheimer's disease. ...Whether the CP is involved in the recruitment of MDMs to the injured brain after ischemic stroke is unknown.
Adult male C57BL/6 mice were subjected to focal cortical ischemia by permanent occlusion of the distal branch of the right middle cerebral artery. Choroid plexus tissues were collected and analyzed for Vcam1, Madcam1, Cx
cl1, Ccl2, Nt5e, and Ifnγ expression at different timepoints after stroke using qPCR. Changes of MDMs in CP and cerebrospinal fluid (CSF) at 1 day and 3 days after stroke were analyzed using flow cytometry. Infiltration of MDMs into CP and CSF were validated using β-actin-GFP chimeric mice and Fgd5-CreERT2 x Lox-stop-lox-Tomato mice. CD115+ monocytes were isolated using a magnetic cell separation system from bone marrow of Cx
cr1-GFP or wild-type C57BL/6 donor mice. The freshly isolated monocytes or M2-like MDMs primed in vitro with IL4 and IL13 were stereotaxically injected into the lateral ventricle of stroke-affected mice to trace for their migration into ischemic hemisphere or to assess their effect on post-stroke recovery using open field, corridor, and active avoidance behavioral tests.
We found that CP responded to cortical stroke by upregulation of gene expression for several possible mediators of MDM trafficking and, concomitantly, MDMs increased in CP and cerebrospinal fluid (CSF). We then confirmed that MDMs infiltrated from blood into CP and CSF after the insult using β-actin-GFP chimeric mice and Fgd5-CreERT2 x Lox-stop-lox-Tomato mice. When MDMs were directly administered into CSF following stroke, they homed to the ischemic hemisphere. If they had been primed in vitro prior to their administration to become M2-like macrophages, they promoted post-stroke recovery of motor and cognitive function without influencing infarct volume.
Our findings suggest the possibility that autologous transplantation of M2-like MDMs into CSF might be developed into a new strategy for promoting recovery also in patients with stroke.
Transplanted neurons derived from stem cells have been proposed to improve function in animal models of human disease by various mechanisms such as neuronal replacement. However, whether the grafted ...neurons receive functional synaptic inputs from the recipient's brain and integrate into host neural circuitry is unknown. Here we studied the synaptic inputs from the host brain to grafted cortical neurons derived from human induced pluripotent stem cells after transplantation into stroke-injured rat cerebral cortex. Using the rabies virus-based trans-synaptic tracing method and immunoelectron microscopy, we demonstrate that the grafted neurons receive direct synaptic inputs from neurons in different host brain areas located in a pattern similar to that of neurons projecting to the corresponding endogenous cortical neurons in the intact brain. Electrophysiological in vivo recordings from the cortical implants show that physiological sensory stimuli, i.e. cutaneous stimulation of nose and paw, can activate or inhibit spontaneous activity in grafted neurons, indicating that at least some of the afferent inputs are functional. In agreement, we find using patch-clamp recordings that a portion of grafted neurons respond to photostimulation of virally transfected, channelrhodopsin-2-expressing thalamo-cortical axons in acute brain slices. The present study demonstrates, for the first time, that the host brain regulates the activity of grafted neurons, providing strong evidence that transplanted human induced pluripotent stem cell-derived cortical neurons can become incorporated into injured cortical circuitry. Our findings support the idea that these neurons could contribute to functional recovery in stroke and other conditions causing neuronal loss in cerebral cortex.
COVID-19 typically manifests as a respiratory illness, but several clinical reports have described gastrointestinal symptoms. This is particularly true in children in whom gastrointestinal symptoms ...are frequent and viral shedding outlasts viral clearance from the respiratory system. These observations raise the question of whether the virus can replicate within the stomach. Here we generate gastric organoids from fetal, pediatric, and adult biopsies as in vitro models of SARS-CoV-2 infection. To facilitate infection, we induce reverse polarity in the gastric organoids. We find that the pediatric and late fetal gastric organoids are susceptible to infection with SARS-CoV-2, while viral replication is significantly lower in undifferentiated organoids of early fetal and adult origin. We demonstrate that adult gastric organoids are more susceptible to infection following differentiation. We perform transcriptomic analysis to reveal a moderate innate antiviral response and a lack of differentially expressed genes belonging to the interferon family. Collectively, we show that the virus can efficiently infect the gastric epithelium, suggesting that the stomach might have an active role in fecal-oral SARS-CoV-2 transmission.
A hallmark of adult hematopoiesis is the continuous replacement of blood cells with limited lifespans. While active hematopoietic stem cell (HSC) contribution to multilineage hematopoiesis is the ...foundation of clinical HSC transplantation, recent reports have questioned the physiological contribution of HSCs to normal/steady-state adult hematopoiesis. Here, we use inducible lineage tracing from genetically marked adult HSCs and reveal robust HSC-derived multilineage hematopoiesis. This commences via defined progenitor cells, but varies substantially in between different hematopoietic lineages. By contrast, adult HSC contribution to hematopoietic cells with proposed fetal origins is neglible. Finally, we establish that the HSC contribution to multilineage hematopoiesis declines with increasing age. Therefore, while HSCs are active contributors to native adult hematopoiesis, it appears that the numerical increase of HSCs is a physiologically relevant compensatory mechanism to account for their reduced differentiation capacity with age.
Human cellular reprogramming to induced pluripotency is still an inefficient process, which has hindered studying the role of critical intermediate stages. Here we take advantage of high efficiency ...reprogramming in microfluidics and temporal multi-omics to identify and resolve distinct sub-populations and their interactions. We perform secretome analysis and single-cell transcriptomics to show functional extrinsic pathways of protein communication between reprogramming sub-populations and the re-shaping of a permissive extracellular environment. We pinpoint the HGF/MET/STAT3 axis as a potent enhancer of reprogramming, which acts via HGF accumulation within the confined system of microfluidics, and in conventional dishes needs to be supplied exogenously to enhance efficiency. Our data suggest that human cellular reprogramming is a transcription factor-driven process that it is deeply dependent on extracellular context and cell population determinants.
Recent advancements in cell engineering have succeeded in manipulating cell identity with the targeted overexpression of specific cell fate determining transcription factors in a process named ...transcriptional programming. Neurogenin2 (NGN2) is sufficient to instruct pluripotent stem cells (PSCs) to acquire a neuronal identity when delivered with an integrating system, which arises some safety concerns for clinical applications. A non-integrating system based on modified messenger RNA (mmRNA) delivery method, represents a valuable alternative to lentiviral-based approaches. The ability of NGN2 mmRNA to instruct PSC fate change has not been thoroughly investigated yet. Here we aimed at understanding whether the use of an NGN2 mmRNA-based approach combined with a miniaturized system, which allows a higher transfection efficiency in a cost-effective system, is able to drive human induced PSCs (hiPSCs) toward the neuronal lineage. We show that NGN2 mRNA alone is able to induce cell fate conversion. Surprisingly, the outcome cell population accounts for multiple phenotypes along the neural development trajectory. We found that this mixed population is mainly constituted by neural stem cells (45% ± 18 PAX6 positive cells) and neurons (38% ± 8 βIIITUBULIN positive cells) only when NGN2 is delivered as mmRNA. On the other hand, when the delivery system is lentiviral-based, both providing a constant expression of NGN2 or only a transient pulse, the outcome differentiated population is formed by a clear majority of neurons (88% ± 1 βIIITUBULIN positive cells). Altogether, our data confirm the ability of NGN2 to induce neuralization in hiPSCs and opens a new point of view in respect to the delivery system method when it comes to transcriptional programming applications.
Induced pluripotent stem cells (iPSCs) or their progeny, derived from human somatic cells, can give rise to functional improvements after intracerebral transplantation in animal models of stroke. ...Previous studies have indicated that reactive gliosis, which is associated with stroke, inhibits neurogenesis from both endogenous and grafted neural stem/progenitor cells (NSPCs) of rodent origin. Here we have assessed whether reactive astrocytes affect the fate of human iPSC-derived NSPCs transplanted into stroke-injured brain. Mice with genetically attenuated reactive gliosis (deficient for GFAP and vimentin) were subjected to cortical stroke and cells were implanted adjacent to the ischemic lesion one week later. At 8 weeks after transplantation, immunohistochemical analysis showed that attenuated reactive gliosis did not affect neurogenesis or commitment towards glial lineage of the grafted NSPCs. Our findings, obtained in a human-to-mouse xenograft experiment, provide evidence that the reactive gliosis in stroke-injured brain does not affect the formation of new neurons from intracortically grafted human iPSC-derived NSPCs. However, for a potential clinical translation of these cells in stroke, it will be important to clarify whether the lack of effect of reactive gliosis on neurogenesis is observed also in a human-to-human experimental setting.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Several neurodegenerative diseases cause loss of cortical neurons, leading to sensory, motor, and cognitive impairments. Studies in different animal models have raised the possibility that ...transplantation of human cortical neuronal progenitors, generated from pluripotent stem cells, might be developed into a novel therapeutic strategy for disorders affecting cerebral cortex. For example, we have shown that human long‐term neuroepithelial‐like stem (lt‐NES) cell‐derived cortical neurons, produced from induced pluripotent stem cells and transplanted into stroke‐injured adult rat cortex, improve neurological deficits and establish both afferent and efferent morphological and functional connections with host cortical neurons. So far, all studies with human pluripotent stem cell‐derived neurons have been carried out using xenotransplantation in animal models. Whether these neurons can integrate also into adult human brain circuitry is unknown. Here, we show that cortically fated lt‐NES cells, which are able to form functional synaptic networks in cell culture, differentiate to mature, layer‐specific cortical neurons when transplanted ex vivo onto organotypic cultures of adult human cortex. The grafted neurons are functional and establish both afferent and efferent synapses with adult human cortical neurons in the slices as evidenced by immuno‐electron microscopy, rabies virus retrograde monosynaptic tracing, and whole‐cell patch‐clamp recordings. Our findings provide the first evidence that pluripotent stem cell‐derived neurons can integrate into adult host neural networks also in a human‐to‐human grafting situation, thereby supporting their potential future clinical use to promote recovery by neuronal replacement in the patient's diseased brain.
Induced pluripotent stem cell-derived (iPS-derived) neural precursor cells may represent the ideal autologous cell source for cell-based therapy to promote remyelination and neuroprotection in myelin ...diseases. So far, the therapeutic potential of reprogrammed cells has been evaluated in neonatal demyelinating models. However, the repair efficacy and safety of these cells has not been well addressed in the demyelinated adult CNS, which has decreased cell plasticity and scarring. Moreover, it is not clear if these induced pluripotent-derived cells have the same reparative capacity as physiologically committed CNS-derived precursors. Here, we performed a side-by-side comparison of CNS-derived and skin-derived neural precursors in culture and following engraftment in murine models of adult spinal cord demyelination. Grafted induced neural precursors exhibited a high capacity for survival, safe integration, migration, and timely differentiation into mature bona fide oligodendrocytes. Moreover, grafted skin-derived neural precursors generated compact myelin around host axons and restored nodes of Ranvier and conduction velocity as efficiently as CNS-derived precursors while outcompeting endogenous cells. Together, these results provide important insights into the biology of reprogrammed cells in adult demyelinating conditions and support use of these cells for regenerative biomedicine of myelin diseases that affect the adult CNS.
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