Multichannel bridges and NSC synergize to enhance axon regeneration, myelination, synaptic reconnection, and recovery after SCI.

Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected the ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery. These data identify a successful strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.

Multichannel bridges and NSC synergize to enhance axon regeneration, myelination, synaptic reconnection, and recovery after SCI.

Regeneration in the injured spinal cord is limited by physical and chemical barriers. Acute implantation of a multichannel poly(lactide-co-glycolide) (PLG) bridge mechanically stabilizes the injury, modulates inflammation, and provides a permissive environment for rapid cellularization and robust axonal regrowth through this otherwise inhibitory milieu. However, without additional intervention, regenerated axons remain largely unmyelinated (<10%), limiting functional repair. While transplanted human neural stem cells (hNSC) myelinate axons after spinal cord injury (SCI), hNSC fate is highly influenced by the SCI inflammatory microenvironment, also limiting functional repair. Accordingly, we investigated the combination of PLG scaffold bridges with hNSC to improve histological and functional outcome after SCI. In vitro, hNSC culture on a PLG scaffold increased oligodendroglial lineage selection after inflammatory challenge. In vivo, acute PLG bridge implantation followed by chronic hNSC transplantation demonstrated a robust capacity of donor human cells to migrate into PLG bridge channels along regenerating axons and integrate into the host spinal cord as myelinating oligodendrocytes and synaptically integrated neurons. Axons that regenerated through the PLG bridge formed synaptic circuits that connected ipsilateral forelimb muscle to contralateral motor cortex. hNSC transplantation significantly enhanced the total number of regenerating and myelinated axons identified within the PLG bridge. Finally, the combination of acute bridge implantation and hNSC transplantation exhibited robust improvement in locomotor recovery vs. control and hNSC transplant alone. These data identify a successful novel strategy to enhance neurorepair through a temporally layered approach using acute bridge implantation and chronic cell transplantation to spare tissue, promote regeneration, and maximize the function of new axonal connections.

Induction of early neural precursors and derivation of tripotent neural stem cells from human pluripotent stem cells under xeno-free conditions.

Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) can differentiate into many cell types and are important for regenerative medicine; however, further work is needed to reliably differentiate hESC and hiPSC into neural-restricted multipotent derivatives or specialized cell types under conditions that are free from animal products. Toward this goal, we tested the transition of hESC and hiPSC lines onto xeno-free (XF) / feeder-free conditions and evaluated XF substrate preference, pluripotency, and karyotype. Critically, XF transitioned H9 hESC, Shef4 hESC, and iPS6-9 retained pluripotency (Oct-4 and NANOG), proliferation (MKI67 and PCNA), and normal karyotype. Subsequently, XF transitioned hESC and hiPSC were induced with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to generate neuralized spheres containing primitive neural precursors, which could differentiate into astrocytes and neurons, but not oligoprogenitors. Further neuralization of spheres via LIF supplementation and attachment selection on CELLstart substrate generated adherent human neural stem cells (hNSC) with normal karyotype and high proliferation potential under XF conditions. Interestingly, adherent hNSC derived from H9, Shef4, and iPS6-9 differentiated into significant numbers of O4+ oligoprogenitors (∼20-30%) with robust proliferation; however, very few GalC+ cells were observed (∼2-4%), indicative of early oligodendrocytic lineage commitment. Overall, these data demonstrate the transition of multiple hESC and hiPSC lines onto XF substrate and media conditions, and a reproducible neuralization method that generated neural derivatives with multipotent cell fate potential and normal karyotype.

Optimization and scale-up of Wharton's jelly-derived mesenchymal stem cells for clinical applications.

MSCs are promising candidates for stem cell therapy and regenerative medicine. Umbilical cord is the easiest obtainable biological source of MSCs and the Wharton's jelly of the umbilical cord is a rich source of fetus-derived stem cells. However, the use of MSCs for therapeutic application is based on their subsequent large-scale in vitro expansion. A fast and efficient protocol for generation of large quantities of MSCs is required to meet the clinical demand and biomedical research needs. Here we have optimized conditions for scaling up of WJ-MSCs. Low seeding density along with basic fibroblast growth factor (bFGF) supplementation in the growth medium, which is DMEM-KO, resulted in propagation of more than 1 x 10(8) cells within a time period of 15 days from a single umbilical cord. The upscaled WJ-MSCs retained their differentiation potential and immunosuppressive capacity. They expressed the typical hMSC surface antigens and the addition of bFGF in the culture medium did not affect the expression levels of HLA-DR and CD 44. A normal karyotype was confirmed in the large-scale expanded WJ-MSCs. Hence, in this study we attempted rapid clinical-scale expansion of WJ-MSCs which would allow these fetus-derived stem cells to be used for various allogeneic cell-based transplantations and tissue engineering.

Increased proliferation and analysis of differential gene expression in human Wharton's jelly-derived mesenchymal stromal cells under hypoxia.

Multipotent mesenchymal stromal cells (MSCs) from Wharton's jelly (WJ) of umbilical cord bear higher proliferation rate and self-renewal capacity than adult tissue-derived MSCs and are a primitive stromal cell population. Stem cell niche or physiological microenvironment plays a crucial role in maintenance of stem cell properties and oxygen concentration is an important component of the stem cell niche. Low oxygen tension or hypoxia is prevalent in the microenvironment of embryonic stem cells and many adult stem cells at early stages of development. Again, in vivo, MSCs are known to home specifically to hypoxic events following tissue injuries. Here we examined the effect of hypoxia on proliferation and in vitro differentiation potential of WJ-MSCs. Under hypoxia, WJ-MSCs exhibited improved proliferative potential while maintaining multi-lineage differentiation potential and surface marker expression. Hypoxic WJ-MSCs expressed higher mRNA levels of hypoxia inducible factors, notch receptors and notch downstream gene HES1. Gene expression profile of WJ-MSCs exposed to hypoxia and normoxia was compared and we identified a differential gene expression pattern where several stem cells markers and early mesodermal/endothelial genes such as DESMIN, CD34, ACTC were upregulated under hypoxia, suggesting that in vitro culturing of WJ-MSCs under hypoxic conditions leads to adoption of a mesodermal/endothelial fate. Thus, we demonstrate for the first time the effect of hypoxia on gene expression and growth kinetics of WJ-MSCs. Finally, although WJ-MSCs do not induce teratomas, under stressful and long-term culture conditions, MSCs can occasionally undergo transformation. Though there were no chromosomal abnormalities, certain transformation markers were upregulated in a few of the samples of WJ-MSCs under hypoxia.

Long-term expansion and pluripotent marker array analysis of Wharton's jelly-derived mesenchymal stem cells.

Mesenchymal stem cells (MSCs) with their multilineage developmental plasticity comprise a promising tool for regenerative cell-based therapy. Despite important biological properties, which the MSCs from different sources share, the differences between them are poorly understood. Hence, it is required to assign a molecular signature to each of these MSC populations, based on stem cell related genes and early lineage or developmental markers. Understanding their propensity to differentiate to different lineages is fundamental for the development of successful cell-based therapies. Culture expansion of MSCs is a prerequisite, since high absolute numbers of stem cells are required to attain a clinical dose. Here, we compared the different culture conditions for long-term expansion of human MSCs isolated from the Wharton's jelly (WJ) of the umbilical cord while preserving their stem cell characteristics and differentiation potential. We find that DMEM-KO and DMEM-F12 are superior as compared to the other media tested in supporting the in vitro expansion of the WJ-MSCs. We studied the gene expression profile of WJ and bone marrow-derived MSCs (BM-MSCs) both at early and late passages using Human Stem Cell Pluripotency Array, and our data revealed differences at the transcriptional level between the two MSC types. Compared to BM-MSCs, WJ-MSCs had higher expression of undifferentiated human embryonic stem cell (hES) markers like NANOG, DNMT3B, and GABRB3, pluripotent/stem cell markers, as well as some early endodermal markers both at early and late passages. To conclude, WJ-MSCs possess properties of true stem cells, which they retain even after extended in vitro culturing.