Regeneration of inner ear cells from stem cell precursors--a future concept of hearing rehabilitation?

The use of stem cells offers new and powerful strategies for future tissue development and engineering. Common features of stem cells are both their capacity for self-renewal and the ability to differentiate into mature effector cells. Since the establishment of embryonic stem cells from early human embryos, research on and clinical application of human ES cells belong to the most controversial topics in our society. Great hopes are based upon the remarkable observation that human ES cells can be greatly expanded in vitro, and that they can differentiate into various clinically important cell types. Recent advances in the cloning of mammals by nuclear transplantation provide new concepts for autologous replacement of damaged and degenerated tissues. In contrast, somatic stem cells of the adult organism were considered to be more restricted in their developmental potential. However, recent investigations suggest that somatic stem cells may have a wider differentiation potential than previously thought. In otology, initial experiments have revealed neural stem cell survival in cochlear cell cultures and under neurotrophin influence, neural stem cells seemed to develop into a neuronal phenotype. Further studies have to be carried out to investigate the full potential of stem cells as well as the molecular mechanisms that are involved in regulating cellular identity and plasticity. Clinically, advances in stem cell biology may provide a permanent source of replacement cells for treating human diseases and could open the development of new concepts for cell and tissue regeneration for a causal treatment of chronic degenerative diseases.     

CNS Targets Support and Sustain Differentiation of Cultured Neuronal and Retinal Progenitor Cells

Superior colliculus (SC) is the target of retinal neurons, allowing them to form connections. Cultured stem cells/progenitors can potentially be used as donor tissue to reconstruct degenerated retina including perhaps replacing lost ganglion cells in glaucoma. In which case, it will be essential for these cells to integrate with the central nervous system targets. Here, we have investigated if the mid-brain region containing superior colliculus (SC) provides a permissive environment for the survival and differentiation of neural progenitors, including retinal progenitor cells propagated in cultures. Neural (NPCs) and retinal progenitor cells (RPCs) from green fluorescent protein (GFP) transgenic mice were cultured. Passage two through four neural and retinal progenitor cells were subsequently cocultured with the SC organotypic slices and maintained in culture for 17 and eight days respectively. Differentiation of the neurons was studied by immunocytochemistry for retinotypic neuronal markers. Retinal progenitor cells cocultured with SC slices were able to differentiate into various neuronal morphologies. Some cocultured progenitor cells differentiated into neurons as suggested by class III β tubulin immunoreactivity. In addition, specific retinotypic neuronal differentiation of RPC was detected by immunoreactivity for calbindin and PKC. SC provides a permissive environment that supports survival and differentiation of the progenitor cells.     

Differentiation of human adipose-derived adult stem cells into neuronal tissue: Does it work?

Adipose tissue contains many cells and proteins that are of value not only for their potential therapeutic applications, but also for the low cost of their harvest and delivery. Mesenchymal stem cells (MSC) were originally isolated from the bone marrow, although similar populations have been isolated from adipose and other tissues. At one time, neural tissues were not regarded as regenerative populations of cells. Therefore, the identification of cell populations capable of neuronal differentiation has generated immense interest. Adipose tissue may represent an alternative source of cells that are capable of neuronal differentiation, potentially enhancing its use in the treatment of neurological disease. The aim of this review is to cover the current state of knowledge of the differentiation potential of human adipose-derived stem (ADAS) cells, specifically their ability to give rise to neuronal cells in vitro. This review presents and discusses different protocols used for inducing human ADAS cells to differentiate in vitro, and the neuronal markers utilized in each system.     

Neuronal stem/progenitor cells in the vertebrate eye

We acquire information from the outside world through our eyes which contain the retina, the photosensitive component of the central nervous system. Once the adult mammalian retina is damaged, the retinal neuronal death causes a severe loss of visual function. It has been believed that the adult mammalian retina had no regenerative capacity. However, the identification of neuronal progenitor cells in the retina sheds some light on cellular therapies for damaged retinal regeneration. In this review, we highlight three potential stem/progenitor cells in the eye, the ciliary body epithelium cells, the iris pigmented epithelium cells, and Müller glia. In order to make them prime candidates for the possible treatment of retinal diseases, it is important to understand their basic characters. In addition, we discuss the key signaling molecules that function extracellularly and determine whether neuronal progenitors remain quiescent, proliferate, or differentiate. Finally, we introduce a secreted protein, Tsukushi, which is a possible candidate as a niche molecule for retinal stem/progenitor cells.     

从胚胎干细胞到视网膜

视网膜退行性病变影响着全世界数百万人。然而,视网膜是人体再生能力很差的一类组织,成年机体无法自我更新那些病变中丢失的视网膜细胞,导致视网膜退行性病变的不可逆性。因此,恢复患者视觉将依赖于引入外源细胞替代丢失的视网膜神经元。胚胎干细胞（ES细胞）具有无限的自我更新能力和形成机体所有类型细胞的巨大分化潜力。这两个特性使得ES细胞成为细胞替代疗法的理想供体细胞。近年来,人们在探索将ES和诱导多能干细胞（iPS细胞）体外定向诱导分化为视网膜神经元,甚至整个视网膜方面已取得多项进展,并且体外形成的视网膜细胞可以与宿主视网膜整合。在此篇综述中,首先简要概括哺乳动物视网膜的组织结构、发育过程和调控机制,然后,重点阐述近年来科研工作者探索ES/iPS细胞体外诱导分化为视网膜细胞和组织的研究进展。     

Isolation of Neural Stem/Progenitor Cells from the Periventricular Region of the Adult Rat and Human Spinal Cord

Adult rat and human spinal cord neural stem/progenitor cells (NSPCs) cultured in growth factor-enriched medium allows for the proliferation of multipotent, self-renewing, and expandable neural stem cells. In serum conditions, these multipotent NSPCs will differentiate, generating neurons, astrocytes, and oligodendrocytes. The harvested tissue is enzymatically dissociated in a papain-EDTA solution and then mechanically dissociated and separated through a discontinuous density gradient to yield a single cell suspension which is plated in neurobasal medium supplemented with epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and heparin. Adult rat spinal cord NSPCs are cultured as free-floating neurospheres and adult human spinal cord NSPCs are grown as adherent cultures. Under these conditions, adult spinal cord NSPCs proliferate, express markers of precursor cells, and can be continuously expanded upon passage. These cells can be studied in vitro in response to various stimuli, and exogenous factors may be used to promote lineage restriction to examine neural stem cell differentiation. Multipotent NSPCs or their progeny can also be transplanted into various animal models to assess regenerative repair.     

In vitro expanded stem cells from the developing retina fail to generate photoreceptors but differentiate into myelinating oligodendrocytes

Cell transplantation to treat retinal degenerative diseases represents an option for the replacement of lost photoreceptor cells. In vitro expandable cells isolated from the developing mammalian retina have been suggested as a potential source for the generation of high numbers of donor photoreceptors. In this study we used standardized culture conditions based on the presence of the mitogens FGF-2 and EGF to generate high numbers of cells in vitro from the developing mouse retina. These presumptive 'retinal stem cells' ('RSCs') can be propagated as monolayer cultures over multiple passages, express markers of undifferentiated neural cells, and generate neuronal and glial cell types upon withdrawal of mitogens in vitro or following transplantation into the adult mouse retina. The proportion of neuronal differentiation can be significantly increased by stepwise removal of mitogens and inhibition of the notch signaling pathway. However, 'RSCs', by contrast to their primary counterparts in vivo, i.e. retinal progenitor cells, loose the expression of retina-specific progenitor markers like Rax and Chx10 after passaging and fail to differentiate into photoreceptors both in vitro or after intraretinal transplantation. Notably, 'RSCs' can be induced to differentiate into myelinating oligodendrocytes, a cell type not generated by primary retinal progenitor cells. Based on these findings we conclude that 'RSCs' expanded in high concentrations of FGF-2 and EGF loose their retinal identity and acquire features of in vitro expandable neural stem-like cells making them an inappropriate cell source for strategies aimed at replacing photoreceptor cells in the degenerated retina.     

Adult stem cells in neural repair: Current options,limitations and perspectives

Stem cells represent a promising step for the future of regenerative medicine. As they are able to differentiate into any cell type, tissue or organ, these cells are great candidates for treatments against the worst diseasesthat defy doctors and researchers around the world. Stem cells can be divided into three main groups:(1) embryonic stem cells;(2) fetal stem cells; and(3) adult stem cells. In terms of their capacity for proliferation, stem cells are also classified as totipotent, pluripotent or multipotent. Adult stem cells, also known as somatic cells, are found in various regions of the adult organism, such as bone marrow, skin, eyes, viscera and brain. They can differentiate into unipotent cells of the residing tissue, generally for the purpose of repair. These cells represent an excellent choice in regenerative medicine, every patient can be a donor of adult stem cells to provide a more customized and efficient therapy against various diseases, in other words, they allow the opportunity of autologous transplantation. But in order to start clinical trials and achieve great results, we need to understand how these cells interact with the host tissue, how they can manipulate or be manipulated by the microenvironment where they will be transplanted and for how long they can maintain their multipotent state to provide a full regeneration.     

Embryonic stem cell-derived neural progenitors as non-tumorigenic source for dopaminergic neurons

AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.     

Umbilical cord blood: a unique source of pluripotent stem cells for regenerative medicine

It is estimated that almost 1 in 3 individuals in the United States might benefit from regenerative medicine therapy. Unfortunately, embryonic stem (ES) cell therapies are currently limited by ethical, political, biological and regulatory hurdles. Thus, for the foreseeable future, the march of regenerative medicine to the clinic will depend upon the development of non-ES cell therapies. Current sources of non-ES cells easily available in large numbers can be found in the bone marrow, adipose tissue and umbilical cord blood. Each of these types of stem cells has already begun to be utilized to treat a variety of diseases. This review will show that cord blood (CB) contains multiple populations of ES-like and other pluripotential stem cells, capable of giving rise to hematopoietic, epithelial, endothelial, and neural tissues both in vitro and in vivo. Cumulatively, the identification and isolation of these populations of pluripotent stem cells within cord blood represents a scientific breakthrough that could potentially impact every field of medicine, via their use in regenerative medicine. Thus, CB stem cells are amenable to treatment of a wide variety of diseases including cardiovascular, hepatic, ophthalmic, orthopaedic, neurological and endocrine diseases.     

Induced pluripotency and direct reprogramming: a new window for treatment of neurodegenerative diseases

Human embryonic stem cells (hESCs) are pluripotent cells that have the ability of unlimited self-renewal and can be differentiated into different cell lineages, including neural stem (NS) cells. Diverse regulatory signaling pathways of neural stem cells differentiation have been discovered, and this will be of great benefit to uncover the mechanisms of neuronal differentiation in vivo and in vitro. However, the limitations of hESCs resource along with the religious and ethical concerns impede the progress of ESCs application. Therefore, the induced pluripotent stem cells (iPSCs) via somatic cell reprogramming have opened up another new territory for regenerative medicine. iPSCs now can be derived from a number of lineages of cells, and are able to differentiate into certain cell types, including neurons. Patient-specific iPSCs are being used in human neurodegenerative disease modeling and drug screening. Furthermore, with the development of somatic direct reprogramming or lineage reprogramming technique, a more effective approach for regenerative medicine could become a complement for iPSCs.     

High-efficient generation of induced pluripotent stem cells from human astrocytes

The reprogramming of human somatic cells to induced pluripotent stem (hiPS) cells enables the possibility of generating patient-specific autologous cells for regenerative medicine. A number of human somatic cell types have been reported to generate hiPS cells, including fibroblasts, keratinocytes and peripheral blood cells, with variable reprogramming efficiencies and kinetics. Here, we show that human astrocytes can also be reprogrammed into hiPS (ASThiPS) cells, with similar efficiencies to keratinocytes, which are currently reported to have one of the highest somatic reprogramming efficiencies. ASThiPS lines were indistinguishable from human embryonic stem (ES) cells based on the expression of pluripotent markers and the ability to differentiate into the three embryonic germ layers in vitro by embryoid body generation and in vivo by teratoma formation after injection into immunodeficient mice. Our data demonstrates that a human differentiated neural cell type can be reprogrammed to pluripotency and is consistent with the universality of the somatic reprogramming procedure.     

BMP signaling regulates the differentiation of mouse embryonic stem cells into lung epithelial cell lineages

Somatic stem/progenitor cells are known to be present in most adult tissues. However, those in the lung have limited abilities for tissue regeneration after serious damage as a result of chronic disease. Therefore, regenerative medicine using exogenous stem cells has been suggested for the treatment of progressive lung diseases such as chronic obstructive pulmonary disease and pulmonary fibrosis. Embryonic stem (ES) cells and induced pluripotent stem cells, with their potent differentiation abilities, are promising sources for the generation of various tissue cells. In this study, we investigated the effects of various differentiation-inducing growth factors on the differentiation of lung cells from ES cells in vitro. Several factors, including activin, nodal, and noggin, significantly promoted the induction of Nkx2.1-positive lung progenitor cells when cells were cultured as embryoid bodies. Bone morphogenetic protein (BMP) 4 signaling controls the lineage commitment of lung cells along the proximal–distal axis. BMP4 promotes the induction of distal cell lineages of alveolar bud, such as Clara cells and mucus-producing goblet cells. These results suggest that several developmentally essential factors, including nodal/activin and BMP signaling, are important in the control of the differentiation of lung epithelial cells from mouse ES cells in vitro.     

Differentiation of embryonic stem cells into retinal neurons

Mouse embryonic stem (ES) cells are continuous cell lines derived from the inner mass of blastocysts. Neural progenitors derived from these cells serve as an excellent model for controlled neural differentiation and as such have tremendous potential to understand and treat neurodegenerative diseases. Here, we demonstrate that ES cell-derived neural progenitors express regulatory factors needed for retinal differentiation and that in response to epigenetic cues a subset of them differentiate along photoreceptor lineage. During the differentiation, they activate photoreceptor regulatory genes, suggesting that ES cell-derived neural progenitors recruit mechanisms normally used for photoreceptor differentiation in vivo. These observations suggest that ES cells can serve as an excellent model for understanding mechanisms that regulate specification of retinal neurons and as an unlimited source of neural progenitors for treating degenerative diseases of the retina by cell replacement.     

Stem‐cell‐based therapies for retinal degenerative diseases: Current challenges in the establishment of new treatment strategies

Various advances have been made in the treatment of retinal diseases, including new treatment strategies and innovations in surgical devices. However, the treatment of degenerative retinal diseases, such as retinitis pigmentosa (RP) and age‐related macular degeneration (AMD), continues to pose a significant challenge. In this review, we focus on the use of embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) to treat retinal diseases by harnessing the ability of stem cells to differentiate into different body tissues. The retina is a tissue specialized for light sensing, and its degradation leads to vision loss. As part of the central nervous system, the retina has very low regenerative capability, and therefore, treatment options are limited once it degenerates. Nevertheless, innovations in methods to induce the generation of retinal cells and tissues from ESCs/iPSCs enable the development of novel approaches for these irreversible diseases. Here we review some historical background and current clinical trials involving the use of stem‐cell‐derived retinal pigment epithelial cells for AMD treatment and stem cell‐derived retinal cells/tissues for RP therapy. Finally, we discuss our future vision of regenerative treatment for retinal diseases with a partial focus on our studies and introduce other interesting approaches for restoring vision.     

Adult neural stem cells: response to stroke injury and potential for therapeutic applications

The plasticity of neural stem/progenitor cells allows a variety of different responses to many environmental cues. In the past decade, significant research has gone into understanding the regulation of neural stem/progenitor cell properties, because of their promise for cell replacement therapies in adult neurological diseases. Both endogenous and grafted neural stem/progenitor cells are known to have the ability to migrate long distances to lesioned sites after brain injury and differentiate into new neurons. Several chemokines and growth factors, including stromal cell-derived factor-1 and vascular endothelial growth factor, have been shown to stimulate the proliferation, differentiation, and migration of neural stem/progenitor cells, and investigators have now begun to identify the critical downstream effectors and signaling mechanisms that regulate these processes. Both our own lab and others have shown that the extracellular matrix and matrix remodeling factors play a critical role in directing cell differentiation and migration of adult neural stem/progenitor cells within injured sites. Identification of these and other molecular pathways involved in stem cell homing into ischemic areas is vital for the development of new treatments. To ensure the best functional recovery, regenerative therapy may require the application of a combination approach that includes cell replacement, trophic support, and neural protection. Here we review the current state of our knowledge about endogenous adult and exogenous neural stem/progenitor cells as potential therapeutic agents for central nervous system injuries.     

Calcium channels and their role in regenerative medicine

Stem cells hold indefinite self-renewable capability that can be differentiated into all desired cell types.Based on their plasticity potential,they are divided into totipotent(morula stage cells),pluripotent(embryonic stem cells),multipotent(hematopoietic stem cells,multipotent adult progenitor stem cells,and mesenchymal stem cells[MSCs]),and unipotent(progenitor cells that differentiate into a single lineage)cells.Though bone marrow is the primary source of multipotent stem cells in adults,other tissues such as adipose tissues,placenta,amniotic fluid,umbilical cord blood,periodontal ligament,and dental pulp also harbor stem cells that can be used for regenerative therapy.In addition,induced pluripotent stem cells also exhibit fundamental properties of self-renewal and differentiation into specialized cells,and thus could be another source for regenerative medicine.Several diseases including neurodegenerative diseases,cardiovascular diseases,autoimmune diseases,virus infection(also coronavirus disease 2019)have limited success with conventional medicine,and stem cell transplantation is assumed to be the best therapy to treat these disorders.Importantly,MSCs,are by far the best for regenerative medicine due to their limited immune modulation and adequate tissue repair.Moreover,MSCs have the potential to migrate towards the damaged area,which is regulated by various factors and signaling processes.Recent studies have shown that extracellular calcium(Ca²⁺)promotes the proliferation of MSCs,and thus can assist in transplantation therapy.Ca²⁺signaling is a highly adaptable intracellular signal that contains several components such as cell-surface receptors,Ca²⁺channels/pumps/exchangers,Ca²⁺buffers,and Ca²⁺sensors,which together are essential for the appropriate functioning of stem cells and thus modulate their proliferative and regenerative capacity,which will be discussed in this review.