Mesenchymal Stem Cell Graft Improves Recovery after Spinal Cord Injury in Adult Rats through Neurotrophic and Pro-Angiogenic Actions

Numerous strategies have been managed to improve functional recovery after spinal cord injury (SCI) but an optimal strategy doesn't exist yet. Actually, it is the complexity of the injured spinal cord pathophysiology that begets the multifactorial approaches assessed to favour tissue protection, axonal regrowth and functional recovery. In this context, it appears that mesenchymal stem cells (MSCs) could take an interesting part. The aim of this study is to graft MSCs after a spinal cord compression injury in adult rat to assess their effect on functional recovery and to highlight their mechanisms of action. We found that in intravenously grafted animals, MSCs induce, as early as 1 week after the graft, an improvement of their open field and grid navigation scores compared to control animals. At the histological analysis of their dissected spinal cord, no MSCs were found within the host despite their BrdU labelling performed before the graft, whatever the delay observed: 7, 14 or 21 days. However, a cytokine array performed on spinal cord extracts 3 days after MSC graft reveals a significant increase of NGF expression in the injured tissue. Also, a significant tissue sparing effect of MSC graft was observed. Finally, we also show that MSCs promote vascularisation, as the density of blood vessels within the lesioned area was higher in grafted rats. In conclusion, we bring here some new evidences that MSCs most likely act throughout their secretions and not via their own integration/differentiation within the host tissue

Induced pluripotent stem cell potential in medicine, specifically focused on reproductive medicine

Since 2006, several laboratories have proved that somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs). iPSCs have enormous potential in stem cell biology as they can give rise to numerous cell lineages, including the three germ layers. In this review, we discuss past and recent advances in human iPSCs used for modeling diseases in vitro, screening drugs to test new treatments, and autologous cell and tissue regenerative therapies, with a special focus on reproductive medicine applications. While this latter field of research is still in its infancy, it holds great promise for investigating germ cell development and studying the genetic and physiopathological mechanisms of infertility. A major cause of infertility is the absence of germ cells in the testes, mainly due to genetic background or as a consequence of gonadotoxic treatments. For these patients, no effective fertility restoration strategy has so far been identified. The derivation of germ cells from iPSCs represents an alternative source of stem cells able to differentiate into spermatozoa. Lessons learned from animal models as well as studies on human iPSCs for reproductive purposes are reviewed

Modeling Klinefelter Syndrome Using Induced Pluripotent Stem Cells Reveals Impaired Germ Cell Differentiation

Klinefelter syndrome (KS), with an incidence between 1/600 and 1/1,000, is the main genetic cause of male infertility. Due to the lack of an accurate study model, the detailed pathogenic mechanisms by which this X chromosome aneuploidy leads to KS features remain unknown. Here, we report the generation and characterization of induced pluripotent stem cells (iPSCs) derived from a patient with KS: 47XXY-iPSCs. In order to compare the potentials of both 47XXY-iPSCs and 46XY-iPSCs to differentiate into the germ cell lineage, we developed a directed differentiation protocol by testing different combinations of factors including bone morphogenetic protein 4 (BMP4), glial-derived neurotrophic factor (GDNF), retinoic acid (RA) and stem cell factor (SCF) for 42 days. Importantly, we found a reduced ability of 47XXY-iPSCs to differentiate into germ cells when compared to 46XY-iPSCs. In particular, upon germ cell differentiation of 47XXY-iPSCs, we found a reduced proportion of cells positive for BOLL, a protein required for germ cell development and spermatogenesis, as well as a reduced proportion of cells positive for MAGEA4, a spermatogonia marker. This reduced ability to generate germ cells was not associated with a decrease of proliferation of 47XXY-iPSC-derived cells but rather with an increase of cell death upon germ cell differentiation as revealed by an increase of LDH release and of capase-3 expression in 47XXY-iPSC-derived cells. Our study supports the idea that 47XXY-iPSCs provides an excellent in vitro model to unravel the pathophysiology and to design potential treatments for KS patients

Stem cells in the adult rat spinal cord: plasticity after injury and treadmill training exercise

ABSTRACT Ependymal cells located around the central canal of the adult spinal cord are considered as a source of neural stem cells (NSCs) and represent an interesting pool of endogenous stem cells for repair strategies. Physical exercise is known to increase ependymal cell proliferation, while improving functional recovery. In this work, we further characterized those endogenous NSCs within the normal and injured adult rat spinal cord and investigated the effects of treadmill training using immunohistochemical and behavioural studies. In uninjured untrained rats, Sox-2, a NSC marker, was detected in all ependymal cells of the central canal, and also scattered throughout the parenchyma of the spinal cord. Within the lesion, Sox-2 expression increased transiently, while the number of nestin-positive ependymal cells increased with a concomitant enhancement of proliferation, as indicated by the mitotic markers Ki67 and BrdU. Exercise, which improved functional recovery and autonomous micturition, maintained nestin expression in both injured and uninjured spinal cords, with a positive correlation between locomotor recovery and the number of nestin-positive cells

Neuroprotection.

<p>MSC transplants favor tissue sparing after SCI. Luxol fast blue/hematoxylin staining on cross sections of MSC-grafted (<b>A</b>) and control injured only (<b>B</b>) rats, 21 days after transplantation. The quantification of spared tissue (<b>C</b>), as assessed by the mean ratio of injured area on total area of the sections, reveals a significant decrease of the lesion extension in MSC treated rats compared to control ones. * p<0.05.</p

Axonal regrowth.

<p>(<b>A</b>–<b>B</b>) GAP43 immunoreactivity on transversal spinal cord sections, 28 days after SCI, in injured + MSC (A) and injured-only (B) groups. Sections were taken at the lesion site. (<b>C</b>) Image analysis doesn't show a significant difference (Student's t-Test, p = 0,23) in the percentage of total lesioned area immunoreactive for GAP43 between treated (2,96 ± 0,35 %) and control (2,25 ± 0,44 %) groups</p

Blood vessel quantification.

<p>(<b>A</b>–<b>B</b>) RECA-1 immunohistological staining on longitudinal sections of MSC-treated (<b>A</b>) and control vehicle-treated (<b>B</b>) spinal cords. Scale bar: 500 µm. (<b>C</b>) Blood vessel quantification within the lesioned site reveals a significant increase in MSC treated rats compared to control-vehicle treated ones. *p<0.05.</p

Behavioral analysis.

<p>(<b>A</b>) Locomotor scores as assessed by the BBB rating scale. MSC grafted rats reach significantly higher scores compared to both control groups. Only MSC transplanted rats reach the weight-supporting step level (score of 9). Injured + MSC vs injured only p = 0.0029; injured + MSC vs injured + vehicle p<0.0001. (<b>B</b>) Grid test scores assessing deficits in descending fine motor control. MSC grafted rats reach higher scores, significantly different from control rats. Injured + MSC vs injured only p<0.0001; injured + MSC vs injured + vehicle p<0.0001.</p

Cytokine array and Elisa results.

<p>(<b>A</b>) Cytokine arrays. Spinal cord extracts from injured vehicle-treated (n = 4) and MSC-treated (n = 5) rats. ß-NGF is significantly increased within the lesioned site 3 days after MSC injection compared to controls. * p<0.05 (mean±S.E.). (<b>B</b>) Histogram showing the amounts, in pg/ml, of the neurotrophins NGF and BDNF as quantified by Elisa, within 2 distinct P12 MSC-conditioned media (M1 and M2). (<b>C</b>) NGF and (<b>D</b>) BDNF fluorescent immunocytochemistry on P12 MSCs <i>in vitro</i>. Scale bar : 50 µm.</p

BrdU immunodetection.

<p>(<b>A</b>) BrdU (FITC)-DAPI immunostaining on MSCs cultured with 1.10⁻⁶M BrdU for 72 h, demonstrating that cells were all labeled before being transplanted. (<b>B–C–D</b>) BrdU (Rhodamine) maintenance in MSCs 3 days (<b>B</b>), 7 days <b>(C)</b> and 21 days (<b>D</b>) after the removal of BrdU from the culture medium. (<b>E</b>) BrdU immunodetection (FITC) on a longitudinal spinal cord tissue section from a rat that received 3 ip BrdU injections after spinal cord injury. Scale bar: 50 µm (A), 100 µm (B, C, D) and 200 µm (E).</p