Katarzyna A. Trzaska

Neurons Derived From Human Mesenchymal Stem Cells Show Synaptic Transmission and Can Be Induced to Produce the Neurotransmitter Substance P by Interleukin‐1α

Mesenchymal stem cells (MSCs) exhibit immune‐suppressive properties, follow a pattern of multilineage differentiation, and exhibit transdifferentiation potential. Ease in expansion from adult bone marrow, as well as its separation from ethical issues, makes MSCs appealing for clinical application. MSCs treated with retinoic acid resulted in synaptic transmission, based on immunostaining of synaptophysin and electrophysiological studies. In situ hybridization indicated that the neurotransmitter gene preprotachykinin‐I was expressed in these cells. However, translation of this gene only occurred after stimulation with interleukin (IL)‐1α. This effect was blunted by costimulation with IL‐1 receptor antagonist. This study reports on the ability of MSCs to be transdifferentiated into neurons with functional synapses with the potential to become polarized towards producing specific neurotransmitters.

Specification of a Dopaminergic Phenotype from Adult Human Mesenchymal Stem Cells

Dopamine (DA) neurons derived from stem cells are a valuable source for cell replacement therapy in Parkinson disease, to study the molecular mechanisms of DA neuron development, and for screening pharmaceutical compounds that target DA disorders. Compared with other stem cells, MSCs derived from the adult human bone marrow (BM) have significant advantages and greater potential for immediate clinical application. We report the identification of in vitro conditions for inducing adult human MSCs into DA cells. Using a cocktail that includes sonic hedgehog and fibroblast growth factors, human BM‐derived MSCs were induced in vitro to become DA cells in 12 days. Based on tyrosine hydroxylase (TH) expression, the efficiency of induction was determined to be ∼67%. The cells develop a neuronal morphology expressing the neuronal markers NeuN and β III tubulin, but not glial markers, glial fibrillary acidic protein and Olig2. As the cells acquire a postmitotic neuronal fate, they downregulate cell cycle activator proteins cyclin B, cyclin‐dependent kinase 2, and proliferating cell nuclear antigen. Molecular characterization revealed the expression of DA‐specific genes such as TH, Pitx3, Nurr1, DA transporter, and vesicular monoamine transporter 2. The induced MSCs also synthesize and secrete DA in a depolarization‐independent manner. The latter observation is consistent with the low expression of voltage gated Na+ and Ca2+ channels in the induced MSCs and suggests that the cells are at an immature stage of development likely representing DA neuronal progenitors. Taken together, the results demonstrate the ability of adult human BM‐derived MSCs to form DA cells in vitro.

Brain‐derived neurotrophic factor facilitates maturation of mesenchymal stem cell‐derived dopamine progenitors to functional neurons

The generation of dopamine (DA) neurons from stem cells holds great promise in the treatment of Parkinson’s disease and other neural disease associated with dysfunction of DA neurons. Mesenchymal stem cells (MSCs) derived from the adult bone marrow show plasticity with regards to generating cells of other germ layers. In addition to reduced ethical concerns, MSCs could be transplanted across allogeneic barriers, making them desirable stem cells for clinical applications. We have reported on the generation of DA cells from human MSCs using sonic hedgehog (SHH), fibroblast growth factor 8 and basic fibroblast growth factor. Despite the secretion of DA, the cells did not show evidence of functional neurons, and were therefore designated DA progenitors. Here, we report on the role of brain‐derived neurotrophic factor (BDNF) in the maturation of the MSC‐derived DA progenitors. 9‐day induced MSCs show significant tropomyosin‐receptor‐kinase B expression, which correlate with its ligand, BDNF, being able to induce functional maturation. The latter was based on Ca2+ imaging analyses and electrophysiology. BDNF‐treated cells showed the following: increases in intracellular Ca2+ upon depolarization and after stimulation with the neurotransmitters acetylcholine and GABA and, post‐synaptic currents by electrophysiological analyses. In addition, BDNF induced increased DA release upon depolarization. Taken together, these results demonstrate the crucial role for BDNF in the functional maturation of MSC‐derived DA progenitors.

Dopaminergic neuronal differentiation protocol for human mesenchymal stem cells.

The generation of dopamine (DA) neurons from stem cells holds great promise for future biomedical research and in the clinical treatment of neurodegenerative diseases, such as Parkinsons disease. Mesenchymal stem cells (MSCs) derived from the adult human bone marrow (BM) can be easily isolated and expanded in culture while maintaining their immense plasticity. Here, we describe a protocol to generate DA-producing cells from adult human MSCs using a cocktail that includes sonic hedgehog (SHH), fibroblast growth factor 8 (FGF8), and basic fibroblast growth factor (bFGF). Electrophysiological functional DA neurons could be achieved by further treatment with brain-derived neurotrophic factor (BDNF). In summary, a protocol is described for the induction of primary BM-derived human MSCs to specific transdifferentiation; in this case, functional DA neurons. The MSC-derived DA cells express DA-specific markers, synthesize, and secrete dopamine. The described method could be used to generate DA cells for various model systems in which DA-producing cells are implicated in pathophysiological conditions.

Loss of RE-1 silencing factor in mesenchymal stem cell-derived dopamine progenitors induces functional maturity

Stem cell-derived dopamine (DA) neurons hold great promise for Parkinsons disease (PD). Mesenchymal stem cells (MSCs) have great potential for clinical applications. The generation of DA cells from MSCs using sonic hedgehog (SHH) and fibroblast growth factors (FGF8 and bFGF) has been reported. However, the DA cells showed weak electrical properties, representing DA neuron progenitors. Since RE-1 Silencing Factor (REST), suppresses mature neuronal genes in neuronal progenitors, we studied its role in the maturation of MSC-derived DA cells. REST expression did not change during the induction process, thus we knocked down REST and subjected MSCs to the same neural induction cocktail. We observed increases in the protein level of the Na(+) voltage-gated channel and tyrosine hydroxylase (TH). Electrophysiological analyses showed spontaneous firings and spontaneous postsynaptic currents, similar to native DA neurons. Taken together, these results show REST as the limiting gene in the generation of functional mature neurons from MSCs.

Current Advances in the Treatment of Parkinsons Disease with Stem Cells

Stem cell replacement has emerged as the novel therapeutic strategy for Parkinsons disease (PD). Control of motor behavior is lost in PD due to the selective degeneration of mesencephalic dopamine neurons (DA) in the substantia nigra. This progressive loss of DA neurons results in devastating symptoms for which there is no cure. Debilitating side effects often result from chronic pharmacological treatment, hence current investigations into cell transplantation therapy as a substitute and/or adjuvant to other therapeutics. Clinical trials with fetal DA tissue have provided evidence that cell transplantation could be a viable alternative. Limited availability of fetal tissue, combined with variable outcome led to emphasis on other sources of cells, such as stem cells. This review focuses on three stem cell sources (embryonic, neural, and adult mesenchymal). Also discussed is the molecular differentiation into mature DA neurons, the various protocols that have been developed to generate DA neurons from various stem cells, and the current state of stem cell therapy for PD.

Neurokinin Receptors as Potential Targets in Breast Cancer Treatment

Despite recent advances in the diagnoses and treatment of breast cancer, this disease continues to be a major cause of death. One of the biggest challenges in breast cancer treatment is bone metastasis. Breast cancer cells (BCCs) are capable of migrating to the bone marrow and utilizing the marrow microenvironment to remain quiescent. While exhibiting quiescence in the marrow, BCCs can evade the effects of conventional cancer treatments such as chemotherapy. Therefore, scientists must find a new paradigm to target these quiescent BCCs. The development of potential targets may require a more comprehensive understanding of the marrow microenvironment and its regulators. The preprotachykinin-1 (PPT-I) gene encodes for the tachykinin peptides, which interact with neurokinin (NK) receptors. Studies have correlated this interaction with BCC integration into the bone marrow and breast cancer progression. In this review, we discuss the roles that different factors of the marrow microenvironment play in breast cancer and targets of NK receptors as potential treatment options.

Neurokinin-A inhibits cell cycle activators in K562 cells and activates Smad 4 through a non-canonical pathway: A novel method in neural–hematopoietic axis

Hematopoiesis is a complexprocess in which blood and immune cells are developed. Among the regulators of hematopoiesis are two members of the G-protein coupled receptor family, neurokinin-1 (NK1) and NK2, which partly encompass the communication between the neural and hematopoietic systems. This communication also involves a complex network of cytokines, and crosstalk between NK1 and NK2. Excessive activation of NK1 has been linked to leukemia. NK2 exerts negative effects on NK1. Previous studies with the hematopoietic progenitor cell line, K562 have identified activated p53 as a mediator of NK2 transcription, which correlated with cell proliferation. This study investigated the mechanism of NK-A mediated inhibition of cell proliferation. K562 was stimulated with 10 nM of NK-A, and the nuclear extracts were analyzed by Westernblots for cell cycle regulators. The studies showed decreases in the cell cycling activators, Cdk2 and Cyclin A, which correlated with increases in p21 and p53. The differentiation protein p19 was unchanged, suggesting that NK-A maintains K562 at cell cycle checkpoints, but does not have roles in differentiation. NK-A appears to regulate TGF-beta 1 production at the level of translation. Despite the production of TGF-beta 1, the activation of Smad 4 occurs by NK-A, via a non-canonical pathway, as indicated by an inhibitor of TGF-beta receptor activators, SB431542. TGF-beta 1 was needed to prevent exacerbated decrease in Cyclin A, but not Cdk2, indicating that it was its role might be limited to balancing the negative regulation of TGF-beta 1. In summary, NK-A enhances translation of TGF-beta 1 in K562 cells. NK-A suppressed cell cycle activators, and activated Smad 4 via a non-canonical pathway, independent of TGF-beta receptor. These findings are significant in the negative regulation of progenitor proliferation, with implications for hematopoiesis and its associated dysfunctions.