Functional Multipotency of Stem Cells: A Conceptual Review of Neurotrophic Factor-Based Evidence and Its Role in Translational Research

We here propose an updated concept of stem cells (SCs), with an emphasis on neural stem cells (NSCs). The conventional view, which has touched principally on the essential property of lineage multipotency (e.g., the ability of NSCs to differentiate into all neural cells), should be broadened to include the emerging recognition of biofunctional multipotency of SCs to mediate systemic homeostasis, evidenced in NSCs in particular by the secretion of neurotrophic factors. Under this new conceptual context and taking the NSC as a leading example, one may begin to appreciate and seek the “logic” behind the wide range of molecular tactics the NSC appears to serve at successive developmental stages as it integrates into and prepares, modifies, and guides the surrounding CNS micro- and macro-environment towards the formation and self-maintenance of a functioning adult nervous system. We suggest that embracing this view of the “multipotency” of the SCs is pivotal for correctly, efficiently, and optimally exploiting stem cell biology for therapeutic applications, including reconstitution of a dysfunctional CNS

Engineering and Selection of Shuffled AAV Genomes: A New Strategy for Producing Targeted Biological Nanoparticles

We report a DNA shuffling–based approach for developing cell type–specific vectors through directed evolution. Capsid genomes of adeno-associated virus (AAV) serotypes 1–9 were randomly fragmented and reassembled using PCR to generate a chimeric capsid library. A single infectious clone (chimeric-1829) containing genome fragments from AAV1, 2, 8, and 9 was isolated from an integrin minus hamster melanoma cell line previously shown to have low permissiveness to AAV. Molecular modeling studies suggest that AAV2 contributes to surface loops at the icosahedral threefold axis of symmetry, while AAV1 and 9 contribute to two- and fivefold symmetry interactions, respectively. The C-terminal domain (AAV9) was identified as a critical structural determinant of melanoma tropism through rational mutagenesis. Chimeric-1829 utilizes heparan sulfate as a primary receptor and transduces melanoma cells more efficiently than all serotypes. Further, chimeric-1829 demonstrates altered tropism in rodent skeletal muscle, liver, and brain including nonhuman primates. We determined a unique immunological profile based on neutralizing antibody (NAb) titer and crossreactivity studies strongly supporting isolation of a synthetic laboratory-derived capsid variant. Application of this technology to alternative cell/tissue types using AAV or other viral capsid sequences is likely to yield a new class of biological nanoparticles as vectors for human gene transfer

Electrophysiological properties of DA induced neurons.

<p>Quantitative analysis of the TH-inductive effects (<b>A</b> & <b>B</b>). The TH-IR neurons were expressed as a percentage of total live cells determined by DAPI staining and also as a percentage of the total number of TuJ1-IR neurons. Results are mean ± S.E.M. of experiments performed three times on independent culture preparations, each performed in duplicate. The TH induction with CM+F+L treatment produced significantly more TH+ cells compared to CM treatment alone (*<i>P</i><0.01) and to non-treated control (**<i>P</i><0.001). (<b>C</b>) The relative abundance of TH and 18 S transcripts was assessed by RT-PCR (see Materials and Methods section for the description of the primers used and the PCR conditions) in control (Ctr), GDNF+bFGF (F)+LIF (L) and in CM+F+L treated cultures (line +) which showed a strong induction of TH in CM+F+L culture condition. (<b>D</b>) Representative current clamp trace of action potentials elicited in response to a 400 pA current injection from a neuron. (<b>E</b>) Example of action potential burst in response to 400 pA current injections. (<b>F</b>) Voltage-activated sodium and potassium currents elicited in response to a −50 mV step. <b>(G)</b> Sample trace of spontaneous EPSCs in a neuron held at −70 mV. (<b>H</b>) Triple labeling immunocytochemical process of hNSC culture used for electrophysiological recording showing the co-localization of TH with biocytin and DAT. Scale bar is 20 µm (H).</p

Human NSCs engraft, extend neurites, and differentiate into TH+ neurons in MPTP-lesioned NHPs.

<p>Coronal section through the midbain showing grafted DA-differentiated hNSCs into the substantia nigra. Two months-post-transplantation, brains were perfused, sectioned, & immunostained for human-specific & TH markers. Arrows indicate two examples of engrafted donor-derived cells, previously transfected with lenti-GFP, coexpressing the nuclear marker DAPI (bleu), the human nuclear marker hNuc (purple), GFP (green) & TH (red). Scale bar is 50 µm.</p

The hNSCs uniformly express markers of neural stem cells.

<p>The isolated hNSCs were passaged every 5–7 days, as described in the Methods section. They grew as a stable homogenous monolayer and uniformly expressed molecular features of NSCs. The top 3 panels: A, B, C represent photomicrographs of undifferentiated hNSCs immunostained with the neural precursor markers nestin (red, A) and vimentin (green, B) and the radial glial marker 3CB2 (purple, C). The cultures were counterstained with the nuclear DNA marker 4′,6-diamidine-2′-phenylindole dihydrochloride (DAPI, blue). The bottom panels (D, E, F) show 7-day old differentiated hNSC cultures processed for indirect immunocytochemistry. Differentiated hNSCs expressed the neuronal-specific marker TuJ1 (green D, E), the astroglia marker GFAP (red, E), the oligodendroglial marker GC (green, F) and DAPI, blue nuclei in all panels. Cell nuclei were stained with live cell marker DAPI (blue, F, G). Scale bars are 20 µm (A–F).</p

Treatment with the glial conditioned media (CM) and growth factors (GF) induce TH expression in hNSCs derived from hESCs.

<p>hNSCs were single cell dissociated in a defined medium and plated on poly-L-ornithine-coated glass coverslips either under control conditions (A) or in glial cell line conditioned media supplemented with the growth factors bFGF (20 ng/ml) and LIF 10 ng/ml for 10 days (B & C). Fixed cultures were stained with nuclear marker DAPI (blue) and processed for TH (red) and for the neuronal marker TuJ1 (green) immunocytochemistry, as described in Materials and Methods. Scale bars are 50 µm (A–C).</p

Grafted dopamine-induced hNSCs maintain their phenotype in MPTP-lesioned NHP.

<p>Coronal section through the caudate/putamen of an MPTP-lesioned NHP (A), show transplant (TX) of hNSCs counterstained with the nuclear marker DAPI (blue) and immunoprocessed for the human-specific cytoplasmic (purple, hCyt) and the TH (red). (B) Photomicrograph taken from the edge of the graft showing triple labeling with hCyt (purple), TH (red) and the synaptic marker synaptophysin (green). An example of TH+ cell is shown in C, and photomicrograph in D show that the neuritic process extended by the hCyt+ grafted cells (purple) do not express the serotoninergic marker (5 HT, green) indicated by the arrow and expressed by endogenous neurons. The TH+ neurite outgrowth express synaptophysin+ puncta (E). Scale bars are 200 µm (A), 20 µm (B–C), 10 µm (D–E).</p

Cellular Repair in the Parkinsonian Nonhuman Primate Brain

Parkinson disease (PD) is a neurodegenerative disorder that provides a useful model for testing cell replacement strategies to rejuvenate the affected dopaminergic neural systems, which have been destroyed by aging and the disease. We first showed that grafts of fetal dopaminergic neurons can reverse parkinsonian motor deficits induced by the toxin, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), validating the feasibility of cellular repair in a primate nervous system. Subsequent clinical trials in Parkinson patients showed encouraging results, including long-term improvement of neurological signs and reduction of medications in some patients. However, many experienced little therapeutic benefit, and some recipients experienced dyskinesias, suggesting a lack of regulated control of the grafts. We have since attempted to improve cell replacements by placing grafts in their correct anatomical location in the substantia nigra and using strategies such as co-grafting fetal striatal tissue or growth factors into the physiologic striatal targets. Moreover, the use of fetal cells depends on a variable supply of donor material, making it difficult to standardize cell quality and quantity. Therefore, we have also explored possibilities of using human neural stem cells (hNSCs) to ameliorate parkinsonism in nonhuman primates with encouraging results. hNSCs implanted into the striatum showed a remarkable migratory ability and were found in the substantia nigra, where a small number appeared to differentiate into dopamine neurons. The majority became growth factor–producing glia that could provide beneficial effects on host dopamine neurons. Studies to determine the optimum stage of differentiation from embryonic stem cells and to derive useful cells from somatic cell sources are in progress