LIF promotes neurogenesis and maintains neural precursors in cell populations derived from spiral ganglion stem cells

BACKGROUND: Stem cells with the ability to form clonal floating colonies (spheres) were recently isolated from the neonatal murine spiral ganglion. To further examine the features of inner ear-derived neural stem cells and their derivatives, we investigated the effects of leukemia inhibitory factor (LIF), a neurokine that has been shown to promote self-renewal of other neural stem cells and to affect neural and glial cell differentiation. RESULTS: LIF-treatment led to a dose-dependent increase of the number of neurons and glial cells in cultures of sphere-derived cells. Based on the detection of developmental and progenitor cell markers that are maintained in LIF-treated cultures and the increase of cycling nestin-positive progenitors, we propose that LIF maintains a pool of neural progenitor cells. We further provide evidence that LIF increases the number of nestin-positive progenitor cells directly in a cell cycle-independent fashion, which we interpret as an acceleration of neurogenesis in sphere-derived progenitors. This effect is further enhanced by an anti-apoptotic action of LIF. Finally, LIF and the neurotrophins BDNF and NT3 additively promote survival of stem cell-derived neurons. CONCLUSION: Our results implicate LIF as a powerful tool to control neural differentiation and maintenance of stem cell-derived murine spiral ganglion neuron precursors. This finding could be relevant in cell replacement studies with animal models featuring spiral ganglion neuron degeneration. The additive effect of the combination of LIF and BDNF/NT3 on stem cell-derived neuronal survival is similar to their effect on primary spiral ganglion neurons, which puts forward spiral ganglion-derived neurospheres as an in vitro model system to study aspects of auditory neuron development.

Robust postmortem survival of murine vestibular and cochlear stem cells

Potential treatment strategies of neurodegenerative and other diseases with stem cells derived from nonembryonic tissues are much less subjected to ethical criticism than embryonic stem cell-based approaches. Here we report the isolation of inner ear stem cells, which may be useful in cell replacement therapies for hearing loss, after protracted postmortem intervals. We found that neonatal murine inner ear tissues, including vestibular and cochlear sensory epithelia, display remarkably robust cellular survival, even 10 days postmortem. Similarly, isolation of sphere-forming stem cells was possible up to 10 days postmortem. We detected no difference in the proliferation and differentiation potential between stem cells isolated directly after death and up to 5 days postmortem. At longer postmortem intervals, we observed that the potency of sphere-derived cells to spontaneously differentiate into mature cell types diminishes prior to the cells losing their potential for self-renewal. Three-week-old mice also displayed sphere-forming stem cells in all inner ear tissues investigated up to 5 days postmortem. In summary, our results demonstrate that postmortem murine inner ear tissue is suited for isolation of stem cells.

Differential distribution of stem cells in the auditory and vestibular organs of the inner ear

The adult mammalian cochlea lacks regenerative capacity, which is the main reason for the permanence of hearing loss. Vestibular organs, in contrast, replace a small number of lost hair cells. The reason for this difference is unknown. In this work we show isolation of sphere-forming stem cells from the early postnatal organ of Corti, vestibular sensory epithelia, the spiral ganglion, and the stria vascularis. Organ of Corti and vestibular sensory epithelial stem cells give rise to cells that express multiple hair cell markers and express functional ion channels reminiscent of nascent hair cells. Spiral ganglion stem cells display features of neural stem cells and can give rise to neurons and glial cell types. We found that the ability for sphere formation in the mouse cochlea decreases about 100-fold during the second and third postnatal weeks; this decrease is substantially faster than the reduction of stem cells in vestibular organs, which maintain their stem cell population also at older ages. Coincidentally, the relative expression of developmental and progenitor cell markers in the cochlea decreases during the first 3 postnatal weeks, which is in sharp contrast to the vestibular system, where expression of progenitor cell markers remains constant or even increases during this period. Our findings indicate that the lack of regenerative capacity in the adult mammalian cochlea is either a result of an early postnatal loss of stem cells or diminishment of stem cell features of maturing cochlear cells.

Chondrogenic potential of human synovial mesenchymal stem cells in alginate

OBJECTIVE: In a recent study, we demonstrated that mesenchymal stem cells (MSCs) derived from the synovial membranes of bovine shoulder joints could differentiate into chondrocytes when cultured in alginate. The purpose of the present study was to establish the conditions under which synovial MSCs derived from aging human donors can be induced to undergo chondrogenic differentiation using the same alginate system. METHODS: MSCs were obtained by digesting the knee-joint synovial membranes of osteoarthritic human donors (aged 59-76 years), and expanded in monolayer cultures. The cells were then seeded at a numerical density of 4x10(6)/ml within discs of 2% alginate, which were cultured in serum-containing or serum-free medium (the latter being supplemented with 1% insulin, transferrin, selenium (ITS). The chondrogenic differentiation capacity of the cells was tested by exposing them to the morphogens transforming growth factor-beta1 (TGF-beta1), TGF-beta2, TGF-beta3, insulin-like growth factor-1 (IGF-1), bone morphogenetic protein-2 (BMP-2) and BMP-7, as well as to the synthetic glucocorticoid dexamethasone. The relative mRNA levels of collagen types I and II, of aggrecan and of Sox9 were determined quantitatively by the real-time polymerase chain reaction (PCR). The extracellular deposition of proteoglycans was evaluated histologically after staining with Toluidine Blue, and that of type-II collagen by immunohistochemistry. RESULTS: BMP-2 induced the chondrogenic differentiation of human synovial MSCs in a dose-dependent manner. The response elicited by BMP-7 was comparable. Both of these agents were more potent than TGF-beta1. A higher level of BMP-2-induced chondrogenic differentiation was achieved in the absence than in the presence of serum. In the presence of dexamethasone, the BMP-2-induced expression of mRNAs for aggrecan and type-II collagen was suppressed; the weaker TGF-beta1-induced expression of these chondrogenic markers was not obviously affected. CONCLUSIONS: We have demonstrated that synovial MSCs derived from the knee joints of aging human donors possess chondrogenic potential. Under serum-free culturing conditions and in the absence of dexamethasone, BMP-2 and BMP-7 were the most potent inducers of this transformation process.

Expression of CD95 on mature leukocytes of MRL/lpr mice after transplantation of genetically modified bone marrow stem cells

Bone marrow transplantation (BMT) is commonly used for the treatment of severe haematological and immunological diseases. For instance, the autoimmune lymphoproliferative syndrome (ALPS) caused by a complete expression defect of CD95 (Fas, APO-1) can be cured by allogeneic BMT. However, since this therapy may not generate satisfactory results when only partially compatible donors are available, we were interested in the development of a potential alternative treatment by using lentiviral gene transfer of a normal copy of CD95 cDNA in hematopoietic stem cells. Here, we show that this approach applied to MRL/lpr mice results in the expression of functional CD95 receptors on the surface of lymphocytes, monocytes, and granulocytes. This suggests that correction of CD95 deficiency can be achieved by gene therapy.

Stem cells as tools in regenerative therapy for retinal degeneration

OBJECTIVE: To describe the use of stem cells (SCs) for regeneration of retinal degenerations. Regenerative medicine intends to provide therapies for severe injuries or chronic diseases where endogenous repair does not sufficiently restore the tissue. Pluripotent SCs, with their capacity to give rise to specialized cells, are the most promising candidates for clinical application. Despite encouraging results, a combination with up-to-date tissue engineering might be critical for ultimate success. DESIGN: The focus is on the use of SCs for regeneration of retinal degenerations. Cell populations include embryonic, neural, and bone marrow-derived SCs, and engineered grafts will also be described. RESULTS: Experimental approaches have successfully replaced damaged photoreceptors and retinal pigment epithelium using endogenous and exogenous SCs. CONCLUSIONS: Stem cells have the potential to significantly impact retinal regeneration. A combination with bioengineering may bear even greater promise. However, ethical and scientific issues have yet to be solved.

A functional model for adult stem cells in epithelial tissues

Tissue turnover, regeneration, and repair take place throughout life. Stem cells are key players in these processes. The characteristics and niches of the stem cell populations in different tissues, and even in related tissues, vary extensively. In this review, stem cell differentiation and stem cell contribution to tissue maintenance and regeneration is compared in the epithelia of the skin, the cornea, the lung, and the intestine. A hierarchical model for adult stem cells is proposed, based on the potency of stem cell subpopulations in a specific tissue. The potency is defined in terms of the maintenance, the repair, and the regeneration of the tissue. The niche supplies cues to maintain the specific stem cell potency.

Isolation of sphere-forming stem cells from the mouse inner ear

The mammalian inner ear has very limited ability to regenerate lost sensory hair cells. This deficiency becomes apparent when hair cell loss leads to hearing loss as a result of either ototoxic insult or the aging process. Coincidently, with this inability to regenerate lost hair cells, the adult cochlea does not appear to harbor cells with a proliferative capacity that could serve as progenitor cells for lost cells. In contrast, adult mammalian vestibular sensory epithelia display a limited ability for hair cell regeneration, and sphere-forming cells with stem cell features can be isolated from the adult murine vestibular system. The neonatal inner ear, however, does harbor sphere-forming stem cells residing in cochlear and vestibular tissues. Here, we provide protocols to isolate sphere-forming stem cells from neonatal vestibular and cochlear sensory epithelia as well as from the spiral ganglion. We further describe procedures for sphere propagation, cell differentiation, and characterization of inner ear cell types derived from spheres. Sphere-forming stem cells from the mouse inner ear are an important tool for the development of cellular replacement strategies of damaged inner ears and are a bona fide progenitor cell source for transplantation studies.