Neuroprotective Effect of Transplanted Human Embryonic Stem Cell-Derived Neural Precursors in an Animal Model of Multiple Sclerosis

BACKGROUND: Multiple sclerosis (MS) is an immune mediated demyelinating disease of the central nervous system (CNS). A potential new therapeutic approach for MS is cell transplantation which may promote remyelination and suppress the inflammatory process. METHODS: We transplanted human embryonic stem cells (hESC)-derived early multipotent neural precursors (NPs) into the brain ventricles of mice induced with experimental autoimmune encephalomyelitis (EAE), the animal model of MS. We studied the effect of the transplanted NPs on the functional and pathological manifestations of the disease. RESULTS: Transplanted hESC-derived NPs significantly reduced the clinical signs of EAE. Histological examination showed migration of the transplanted NPs to the host white matter, however, differentiation to mature oligodendrocytes and remyelination were negligible. Time course analysis of the evolution and progression of CNS inflammation and tissue injury showed an attenuation of the inflammatory process in transplanted animals, which was correlated with the reduction of both axonal damage and demyelination. Co-culture experiments showed that hESC-derived NPs inhibited the activation and proliferation of lymph node-derived T cells in response to nonspecific polyclonal stimuli. CONCLUSIONS: The therapeutic effect of transplantation was not related to graft or host remyelination but was mediated by an immunosuppressive neuroprotective mechanism. The attenuation of EAE by hESC-derived NPs, demonstrated here, may serve as the first step towards further developments of hESC for cell therapy in MS

Enriched Population of PNS Neurons Derived from Human Embryonic Stem Cells as a Platform for Studying Peripheral Neuropathies

BACKGROUND: The absence of a suitable cellular model is a major obstacle for the study of peripheral neuropathies. Human embryonic stem cells hold the potential to be differentiated into peripheral neurons which makes them a suitable candidate for this purpose. However, so far the potential of hESC to differentiate into derivatives of the peripheral nervous system (PNS) was not investigated enough and in particular, the few trials conducted resulted in low yields of PNS neurons. Here we describe a novel hESC differentiation method to produce enriched populations of PNS mature neurons. By plating 8 weeks hESC derived neural progenitors (hESC-NPs) on laminin for two weeks in a defined medium, we demonstrate that over 70% of the resulting neurons express PNS markers and 30% of these cells are sensory neurons. METHODS/FINDINGS: Our method shows that the hNPs express neuronal crest lineage markers in a temporal manner, and by plating 8 weeks hESC-NPs into laminin coated dishes these hNPs were promoted to differentiate and give rise to homogeneous PNS neuronal populations, expressing several PNS lineage-specific markers. Importantly, these cultures produced functional neurons with electrophysiological activities typical of mature neurons. Moreover, supporting this physiological capacity implantation of 8 weeks old hESC-NPs into the neural tube of chick embryos also produced human neurons expressing specific PNS markers in vivo in just a few days. Having the enriched PNS differentiation system in hand, we show for the first time in human PNS neurons the expression of IKAP/hELP1 protein, where a splicing mutation on the gene encoding this protein causes the peripheral neuropathy Familial Dysautonomia. CONCLUSIONS/SIGNIFICANCE: We conclude that this differentiation system to produce high numbers of human PNS neurons will be useful for studying PNS related neuropathies and for developing future drug screening applications for these diseases

Screening ethnically diverse human embryonic stem cells identifies a chromosome 20 minimal amplicon conferring growth advantage

The International Stem Cell Initiative analyzed 125 human embryonic stem (ES) cell lines and 11 induced pluripotent stem (iPS) cell lines, from 38 laboratories worldwide, for genetic changes occurring during culture. Most lines were analyzed at an early and late passage. Single-nucleotide polymorphism (SNP) analysis revealed that they included representatives of most major ethnic groups. Most lines remained karyotypically normal, but there was a progressive tendency to acquire changes on prolonged culture, commonly affecting chromosomes 1, 12, 17 and 20. DNA methylation patterns changed haphazardly with no link to time in culture. Structural variants, determined from the SNP arrays, also appeared sporadically. No common variants related to culture were observed on chromosomes 1, 12 and 17, but a minimal amplicon in chromosome 20q11.21, including three genes expressed in human ES cells, ID1, BCL2L1 and HM13, occurred in >20% of the lines. Of these genes, BCL2L1 is a strong candidate for driving culture adaptation of ES cells

Systemically transplanted mesenchymal stem cells induce vascular-like structure formation in a rat model of vaginal injury.

The beneficial effect of mesenchymal stem cells (MSCs) on wound healing is mostly attributed to a trophic effect that promotes angiogenesis. Whether MSCs can contribute to the formation of new blood vessels by direct differentiation is still controversial. Pelvic floor dysfunction (PFD) is a group of disorders that negatively affect the quality of women's lives. Traditional vaginal surgical repair provides disappointing anatomical outcome. Stem cell transplantation may be used to supplement surgery and improve its outcome. Here we aimed to examine the engraftment, survival, differentiation and angiogenic effect of transplanted MSCs in a vaginal injury rat model. MSCs were obtained from the bone marrow of Sprague Drawley (SD) rats, expanded and characterized in vitro. The MSCs expressed CD90 and CD29, did not express CD45, CD34, CD11b and CD31 and could differentiate into osteogenic, chondrogenic and adipogenic lineages. Cells were labeled with either PKH-26 or GFP and transplanted systemically or locally to female SD rats, just after a standardized vaginal incision was made. Engraftment after local transplantation was less efficient at all-time points compared to systemic administration. In the systemically transplanted animal group, MSCs migrated to the injury site and were present in the healed vagina for at least 30 days. Both systemic and local MSCs transplantation promoted host angiogenesis. Systemically transplanted MSCs created new vascular-like structures by direct differentiation into endothelium. These findings pave the way to further studies of the potential role of MSCs transplantation in improving surgical outcome in women with PFD

Standardization of the Teratoma Assay for Analysis of Pluripotency of Human ES Cells and Biosafety of Their Differentiated Progeny

<div><p>Teratoma tumor formation is an essential criterion in determining the pluripotency of human pluripotent stem cells. However, currently there is no consistent protocol for assessment of teratoma forming ability. Here we present detailed characterization of a teratoma assay that is based on subcutaneous co-transplantation of defined numbers of undifferentiated human embryonic stem cells (hESCs) with mitotically inactivated feeder cells and Matrigel into immunodeficient mice. The assay was highly reproducible and 100% efficient when 100,000 hESCs were transplanted. It was sensitive, promoting teratoma formation after transplantation of 100 hESCs, though larger numbers of animals and longer follow-up were required. The assay could detect residual teratoma forming cells within differentiated hESC populations however its sensitivity was decreased in the presence of differentiated cells. Our data lay the foundation, for standardization of a teratoma assay for pluripotency analysis. The assay can also be used for bio-safety analysis of pluripotent stem cell-derived differentiated progeny.</p> </div

Proposed protocol for a standardized teratoma assay of pluripotency.

<p>The scheme describes our recommendations for the performance and analysis of a standardized teratoma assay of pluripotency.</p

Kinetics of Teratoma Formation after Transplantation of Various Specific Numbers of Undifferentiated HES-1 Cells.

<p>Specific numbers of undifferentiated HES-1 cells were mixed with MMC-treated foreskin fibroblasts (to a total of 1×10⁶ cells) and Matrigel, and transplanted s.c. into NOD/SCID mice. The transplanted animals were monitored weekly for the appearance of tumors, and for the progression of tumor size. The endpoint of the experiments was when the tumors reached a size of ≥ 1 cm³ or 30 weeks after transplantation. (<b>A</b>): Efficiencies of teratoma tumor formation after transplantation of decreasing numbers of undifferentiated HES-1 cells. The trendline is depicted in dotted line. (<b>B</b>): A Kaplan-Meier plot showing the percentage of surviving mice transplanted with decreasing numbers of hESCs, at various time-points (1 W–30 W) during the transplantation experiments. The mice were sacrificed when the tumors reached a volume of ≥ 1 cm³. (<b>C</b>): The average time interval between transplantation and the detection of tumors. (<b>D</b>): The average volume of the teratomas at the time of animal sacrifice. All data relate to animals that developed teratomas. Data presented as mean ± SEM.</p

Histological Analysis of Teratomas Formed after Transplantation of undifferentiated HES-1 Cells.

<p>Images of sections of teratomas, formed after s.c. transplantation of undifferentiated hESCs with their feeders and Matrigel into NOD/SCID mice. (<b>A–C</b>): A teratoma formed from transplantation of 5×10⁵ hESCs. (A) A lower magnification (× 4) of a section of a teratoma showing derivatives of all three germ layers. (B–C) A higher magnification (× 20) of regions of (A) showing ectodermal, and endodermal structures (B) and mesodermal and endodermal structures (C). (<b>D–G</b>): A teratoma formed from transplantation of 100 hESCs. (D) A lower magnification (× 1.25) of a section of a teratoma showing derivatives of all three germ layers (E–G) a higher magnification (× 20) of regions of (D) showing ectodermal structures (E), mesodermal structures (F) and endodermal structures (G).</p

Histological Analysis of Teratomas formed after Transplantation of RPE Cells Spiked with Undifferentiated HES-1 Cells.

<p>(<b>A–B</b>): H&E staining of a section of a teratoma and an RPE graft generated after transplantation of 5×10⁵ HES-1-derived RPE cells spiked with 1×10⁴ undifferentiated HES-1 cells. (<b>C</b>): Efficiency of teratoma formation after transplantation of 5×10⁵ HES-1-derived RPE cells spiked with decreasing numbers of undifferentiated HES-1 cells.</p