The Isolation, Differentiation, and Survival In Vivo of Multipotent Cells from the Postnatal Rat filum terminale

Neural stem cells (NSCs) are undifferentiated cells in the central nervous system (CNS) that are capable of self-renewal and can be induced to differentiate into neurons and glia. Current sources of mammalian NSCs are confined to regions of the CNS that are critical to normal function and surgically difficult to access, which limits their therapeutic potential in human disease. We have found that the filum terminale (FT), a previously unexplored, expendable, and easily accessible tissue at the caudal end of the spinal cord, is a source of multipotent cells in postnatal rats and humans. In this study, we used a rat model to isolate and characterize the potential of these cells. Neurospheres derived from the rat FT are amenable to in vitro expansion in the presence of a combination of growth factors. These proliferating, FT-derived cells formed neurospheres that could be induced to differentiate into neural progenitor cells, neurons, astrocytes, and oligodendrocytes by exposure to serum and/or adhesive substrates. Through directed differentiation using sonic hedgehog and retinoic acid in combination with various neurotrophic factors, FT-derived neurospheres generated motor neurons that were capable of forming neuromuscular junctions in vitro. In addition, FT-derived progenitors that were injected into chick embryos survived and could differentiate into both neurons and glia in vivo

First Report of Circulating MicroRNAs in Tumour Necrosis Factor Receptor-Associated Periodic Syndrome (TRAPS)

Tumor necrosis factor-receptor associated periodic syndrome (TRAPS) is a rare autosomal dominant autoinflammatory disorder characterized by recurrent episodes of long-lasting fever and inflammation in different regions of the body, such as the musculo-skeletal system, skin, gastrointestinal tract, serosal membranes and eye. Our aims were to evaluate circulating microRNAs (miRNAs) levels in patients with TRAPS, in comparison to controls without inflammatory diseases, and to correlate their levels with parameters of disease activity and/or disease severity. Expression levels of circulating miRNAs were measured by Agilent microarrays in 29 serum samples from 15 TRAPS patients carrying mutations known to be associated with high disease penetrance and from 8 controls without inflammatory diseases. Differentially expressed and clinically relevant miRNAs were detected using GeneSpring GX software. We identified a 6 miRNAs signature able to discriminate TRAPS from controls. Moreover, 4 miRNAs were differentially expressed between patients treated with the interleukin (IL)-1 receptor antagonist, anakinra, and untreated patients. Of these, miR-92a-3p and miR-150-3p expression was found to be significantly reduced in untreated patients, while their expression levels were similar to controls in samples obtained during anakinra treatment. MiR-92b levels were inversely correlated with the number of fever attacks/year during the 1st year from the index attack of TRAPS, while miR-377-5p levels were positively correlated with serum amyloid A (SAA) circulating levels. Our data suggest that serum miRNA levels show a baseline pattern in TRAPS, and may serve as potential markers of response to therapeutic intervention

Characterization of the Filum terminale as a neural progenitor cell niche in both rats and humans

Abstract Neural stem cells (NSCs) reside in a unique microenvironment within the central nervous system (CNS) called the NSC niche. Although they are relatively rare, niches have been previously characterized in both the brain and spinal cord of adult animals. Recently, another potential NSC niche has been identified in the filum terminale (FT), which is a thin band of tissue at the caudal end of the spinal cord. While previous studies have demonstrated that NSCs can be isolated from the FT, the in vivo architecture of this tissue and its relation to other NSC niches in the CNS has not yet been established. In this article we report a histological analysis of the FT NSC niche in postnatal rats and humans. Immunohistochemical characterization reveals that the FT is mitotically active and its cells express similar markers to those in other CNS niches. In addition, the organization of the FT most closely resembles that of the adult spinal cord niche. J. Comp. Neurol. 525:661–675, 2017. © 2016 Wiley Periodicals, Inc

Conditions for FT-derived neurosphere differentiation.

<p>Seven different conditions involving various combinations of an adhesive substrate with serum-containing medium were used to differentiate FT-derived neurospheres. After 7 days, the differentiated cells were subsequently immunostained for markers to identify neurons (βTIII, neurofilament), astrocytes (GFAP), and oligodendrocytes (O1). Although all seven conditions generated these cell types, exposing neurospheres to serum resulted in a more rapid differentiation process. N.D.: not done.</p

Generation of motor neurons (MNs) from FT-derived neurospheres.

<p>Individual neurospheres were treated with RA and either Shh-N or Hh-Ag1.3 for 4–5 days, plated onto an adhesive substrate, and cultured in serum-containing medium with appropriate growth factors for 7–10 days. Differentiated cells were subsequently evaluated for the expression of MN and MN progenitor markers. <b>a</b>) Differentiated cells from a P7 FT (30 DIV) stained positive for Olig2 (i, red) Pax6 (ii, green). The merged images co-stained for DAPI (blue) are shown in (iii). <b>b</b>) The MN-specific marker MNR2 is expressed by FT-derived neurospheres (green). Donor: P7 FT, 30 DIV. <b>c</b>) Expression of Tuj-1 (i, green) and ChAT (ii, red). The merged image co-stained for DAPI is shown in (iii). Donor: P6 FT, 36 DIV. Scale bars: 100 µm.</p

<i>In vivo</i> survival and differentiation of FT-derived NPCs transplanted into the developing chick spinal cord.

<p><b>a</b>) Neurospheres were isolated from a P2 transgenic rat that expressed eGFP in every cell. Scale bar: 100 µm. <b>b</b>) GFP⁺ NPCs from a P2 rat were transplanted into a stage 10 (33 hrs) chick neural tube at the prospective rostral end of the developing spinal cord. This image was taken immediately after injection, and the rostral end is pointing towards the bottom. Scale bar: 200 µm. <b>c</b>) After 3 DIO (∼stage 22), GFP⁺ cells had survived and begun to take on the morphology of neurons and glia. Scale bar: 50 µm. <b>d–e</b>) Neurospheres from a 6-month-old human were transplanted into a stage 10 (33 hrs) chick spinal cord at the rostral end. After 3 DIO, transplanted cells were labeled with the human-specific antibody HSP27 (green) and can be seen migrating away from the transplantation site. Two different embryos are shown in (d) and (e). The image in (e)i is a magnified view of the migrating cells from the highlighted area in (e). Scale bars: (d) and (e): 200 µm, (e)i: 50 µm. <b>f–h</b>) After 7 DIO, some FT-derived NPCs (green, f–h(i)) expressed the neuronal marker Tuj-1 (red, f(ii)), the astrocyte marker GFAP (red, g(ii)), or the oligodendrocyte marker MBP (red, h(ii)). In all cases (f–h), a merged image of (i) and (ii) along with the nuclear marker DAPI is shown in (iii). Scale bars: 50 µm.</p

Variable expression of MNR2 in FT-derived neurospheres undergoing MN differentiation.

<p>Scatter graph illustrating the variability in MNR2 expression in differentiated neurospheres under four sets of differentiation conditions. The X axis is the experiment number, and the Y axis is the average proportion of cells derived from each neurosphere expressing MNR2, Tuj-1 and/or GFAP. MNR2, which was used to identify MN generation, was expressed by cells in roughly the same proportion among conditions (1), (2), and (3) with more inconsistent results under condition (4). In 3 of the 28 experiments where the small molecule agonist, Hh-Ag1.3, was used in place of Shh-N, 100% expression of MNR2 was observed. See Results for more detail.</p

Inducing differentiation of FT-derived neurospheres.

<p><b>a</b>) An <b>i</b>ndividual neurosphere from a P5 FT (42 DIV) was plated at T = 0 on a laminin-coated coverslip and cultured in media that contained 10% serum. <b>b</b>) Morphological properties of differentiation were evident after 18 hours of exposure to these differentiating conditions. Scale bar: 100 µm.</p

Developmental history of the FT.

<p><b>a</b>) Illustration of two stages of a human embryo by Streeter (1919) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065974#pone.0065974-Streeter1" target="_blank">[33]</a>, which demonstrates the de-differentiation of the caudal spinal cord into the filum terminale (FT) following the re-absorption of the vestigial tail. The numbers below the drawing indicate the length of the embryo. <b>b</b>) Drawings by Kunitomo (1918) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0065974#pone.0065974-Kunitomo1" target="_blank">[36]</a>of the process of re-absorption of the embryonic tail. The numbers below the illustrations indicate the age in weeks of the fetus, and the numbers above represent the length in millimeters.</p