A Spacetime Odyssey of Neural Progenitors to Generate Neuronal Diversity / 神经科学通报·英文版

To understand how the nervous system develops from a small pool of progenitors during early embryonic development, it is fundamentally important to identify the diversity of neuronal subtypes, decode the origin of neuronal diversity, and uncover the principles governing neuronal specification across different regions. Recent single-cell analyses have systematically identified neuronal diversity at unprecedented scale and speed, leaving the deconstruction of spatiotemporal mechanisms for generating neuronal diversity an imperative and paramount challenge. In this review, we highlight three distinct strategies deployed by neural progenitors to produce diverse neuronal subtypes, including predetermined, stochastic, and cascade diversifying models, and elaborate how these strategies are implemented in distinct regions such as the neocortex, spinal cord, retina, and hypothalamus. Importantly, the identity of neural progenitors is defined by their spatial position and temporal patterning factors, and each type of progenitor cell gives rise to distinguishable cohorts of neuronal subtypes. Microenvironmental cues, spontaneous activity, and connectional pattern further reshape and diversify the fate of unspecialized neurons in particular regions. The illumination of how neuronal diversity is generated will pave the way for producing specific brain organoids to model human disease and desired neuronal subtypes for cell therapy, as well as understanding the organization of functional neural circuits and the evolution of the nervous system.

Engraftment of human induced pluripotent stem cell-derived myogenic progenitors restores dystrophin in mice with duchenne muscular dystrophy

BACKGROUND: Duchenne muscular dystrophy (DMD) is a devastating genetic muscular disorder with no effective treatment that is caused by the loss of dystrophin. Human induced pluripotent stem cells (hiPSCs) offer a promising unlimited resource for cell-based therapies of muscular dystrophy. However, their clinical applications are hindered by inefficient myogenic differentiation, and moreover, the engraftment of non-transgene hiPSC-derived myogenic progenitors has not been examined in the mdx mouse model of DMD. METHODS: We investigated the muscle regenerative potential of myogenic progenitors derived from hiPSCs in mdx mice. The hiPSCs were transfected with enhanced green fluorescent protein (EGFP) vector and defined as EGFP hiPSCs. Myogenic differentiation was performed on EGFP hiPSCs with supplementary of basic fibroblast growth factor, forskolin, 6-bromoindirubin-3'-oxime as well as horse serum. EGFP hiPSCs-derived myogenic progenitors were engrafted into mdx mice via both intramuscular and intravenous injection. The restoration of dystrophin expression, the ratio of central nuclear myofibers, and the transplanted cells-derived satellite cells were accessed after intramuscular and systemic transplantation. RESULTS: We report that abundant myogenic progenitors can be generated from hiPSCs after treatment with these three small molecules, with consequent terminal differentiation giving rise to mature myotubes in vitro. Upon intramuscular or systemic transplantation into mdx mice, these myogenic progenitors engrafted and contributed to human-derived myofiber regeneration in host muscles, restored dystrophin expression, ameliorated pathological lesions, and seeded the satellite cell compartment in dystrophic muscles. CONCLUSIONS: This study demonstrates the muscle regeneration potential of myogenic progenitors derived from hiPSCs using non-transgenic induction methods. Engraftment of hiPSC-derived myogenic progenitors could be a potential future therapeutic strategy to treat DMD in a clinical setting.

Cardiac Cells in Regenerative Processes in Patients with Heart Failure Due to Ischaemic Heart Disease.

Aims: This study aimed to find the cardiac cells which can participate in the processes of regeneration at patients with heart failure due to ischaemic heart disease. To investigate the participation of myosin activating protein kinases in sarcomerogenesis, because sarcomerogenesis is the crucial part of cardiomyocyte differentiation process. Study Design: Resident cardiomyocyte progenitors and dedifferentiated cardiomyocytes were found in left atrial appendages from patients with heart failure due to ischaemic heart disease. We used a cell model of fetal cardiomyocytes with the disassembly contractile apparatus to study the forming of new myofibrils (or sarcomerogenesis) regulated by myosin activating protein kinases. Place and Duration of Study: Cardiology Research and Production Center, Research Center for Obstetrics, Gynecology and Perinatology, Department of Fundamental and Applied Neurobiology of V. Serbsky Federal Medical Research Centre of Psychiatry and Narcology between June 2014 and October 2015. Methodology: We included 10 patients with heart failure due to ischaemic heart disease. Resident cardiomyocyte progenitors and dedifferentiated cardiomyocytes were found by the immunofluorescence approach and the electron microscopy. To determine the myosin activating protein kinases localization in human fetal cardiomyocytes at the 8-9 week heart gestation stage immunofluorescence approach was used. Results: We detected the cardiomyocyte progenitor cells which express c-Kit and Nkx-2.5, other cells express Mdr-1 and GATA-4. Dedifferentiated cardiomyocytes were found. It has been established that smooth muscle, nonmuscle and skeletal myosin light chain kinases are colocalized with nonmuscle myosin in premyofibrils in fetal human cardiomyocytes. Conclusion: We demonstrated that the heart of patients with heart failure due to ischaemic heart disease contains the progenitor resident cardiomyocytes and dedifferentiated cardiomyocytes. These cardiac cells possibly can proliferate and differentiate to mature cardiomyocytes and recover heart function and structure after injury. Myosin activating protein kinases may contribute in myofibril formation during the cardiomyocyte differentiation.

Recent advance in brown adipose physiology and its therapeutic potential

Brown adipose tissue (BAT) is a specialized thermoregulatory organ that has a critical role in the regulation of energy metabolism. Specifically, energy expenditure can be enhanced by the activation of BAT function and the induction of a BAT-like catabolic phenotype in white adipose tissue (WAT). Since the recent recognition of metabolically active BAT in adult humans, BAT has been extensively studied as one of the most promising targets identified for treating obesity and its related disorders. In this review, we summarize information on the developmental origin of BAT and the progenitors of brown adipocytes in WAT. We explore the transcriptional control of brown adipocyte differentiation during classical BAT development and in WAT browning. We also discuss the neuronal control of BAT activity and summarize the recently identified non-canonical stimulators of BAT that can act independently of beta-adrenergic stimulation. Finally, we review new findings on the beneficial effects of BAT activation and development with respect to improving metabolic profiles. We highlight the therapeutic potential of BAT and its future prospects, including pharmacological intervention and cell-based therapies designed to enhance BAT activity and development.

Pluripotent stem cells for cardiac regeneration: Overview of recent advances & emerging trends.

Cell based regenerative therapy has emerged as one of the most promising options of treatment for patients suffering from heart failure. Various adult stem cells types have undergone extensive clinical trials with limited success which is believed to be more of a cytokine effect rather than cell therapy. Pluripotent human embryonic stem cells (hESCs) have emerged as an attractive candidate stem cell source for obtaining cardiomyocytes (CMs) because of their tremendous capacity for expansion and unquestioned potential to differentiate into CMs. Studies carried out in animal models indicate that ES-derived CMs can partially remuscularize infarcted hearts and improve contractile function; however, the effect was not sustained over long follow up periods due to their limited capacity of cell division in vivo. Thus, the concept of transplanting multipotent cardiovascular progenitors derived from ES cells has emerged since the progenitors retain robust proliferative ability and multipotent nature enabling repopulation of other myocardial elements also in addition to CMs. Transplantation of CMs (progenitors) seeded in biodegradable scaffold and gel based engineered constructs has met with modest success due to issues like cell penetration, nutrient and oxygen availability and inflammation triggered during scaffold degradation inversely affecting the seeded cells. Recently cell sheet based tissue engineering involving culturing cells on ‘intelligent’ polymers has been evolved. Generation of a 3-D pulsatile myocardial tissue has been achieved. However, these advances have to be looked at with cautious optimism as many challenges need to be overcome before using these in clinical practice.

In vitro inhibitory effects of imatinib mesylate on stromal cells and hematopoietic progenitors from bone marrow

Imatinib mesylate (IM) is used to treat chronic myeloid leukemia (CML) because it selectively inhibits tyrosine kinase, which is a hallmark of CML oncogenesis. Recent studies have shown that IM inhibits the growth of several non-malignant hematopoietic and fibroblast cells from bone marrow (BM). The aim of the present study was to evaluate the effects of IM on stromal and hematopoietic progenitor cells, specifically in the colony-forming units of granulocyte/macrophage (CFU-GM), using BM cultures from 108 1.5- to 2-month-old healthy Swiss mice. The results showed that low concentrations of IM (1.25 µM) reduced the growth of CFU-GM in clonogenic assays. In culture assays with stromal cells, fibroblast proliferation and α-SMA expression by immunocytochemistry analysis were also reduced in a concentration-dependent manner, with a survival rate of approximately 50% with a dose of 2.5 µM. Cell viability and morphology were analyzed using MTT and staining with acrydine orange/ethidium bromide. Most cells were found to be viable after treatment with 5 µM IM, although there was gradual growth inhibition of fibroblastic cells while the number of round cells (macrophage-like cells) increased. At higher concentrations (15 µM), the majority of cells were apoptotic and cell growth ceased completely. Oil red staining revealed the presence of adipocytes only in untreated cells (control). Cell cycle analysis of stromal cells by flow cytometry showed a blockade at the G0/G1 phases in groups treated with 5-15 µM. These results suggest that IM differentially inhibits the survival of different types of BM cells since toxic effects were achieved.

Systemic injection of recombinant human erythropoietin after focal cerebral ischemia enhances oligodendroglial and endothelial progenitor cells in rat brain / 대한해부학회지

Erythropoietin (EPO) has been demonstrated the ability of recombinant human erythropoietin (r-Hu-EPO), when administered intracerebro-ventricularly, to improve stroke outcome through the reduction of stroke damage. In a brain ischemic model, however, systemic administration of r-Hu-EPO has not been intensely investigated given that in general, large glycosylated molecules have been deemed incapable of crossing the blood-brain barrier. In this study, administration of r-Hu-EPO for 4 days, intraperitoneally after ischemia-reperfusion (I-R) increased the number of bromodeoxyuridine (BrdU)-positive cells in the penumbra (10.1+/-1.4, n=5, P<0.05) and in the subventricular zone (SVZ) of the lateral ventricle (LV) (25+/-2.7, n=5, P<0.05) as compared with those of I-R (penumbra: 2.5+/-0.7; SVZ of LV: 3.8+/-1.5). A significant increase of BrdU-positive cells in these areas was coincident with a strong immunoreactivity of oligodendrocyte progenitor cell marker (2', 3'-cyclic nucleotide 3'-phosphodiesterase). Furthermore, r-Hu-EPO administration increased the number of BrdU-positive cells in the choroid plexus (7.8+/-2.3, n=5, P<0.05) and in cerebral blood vessels (3.5+/-1.3, n=5, P<0.05) when compared with those of I-R (choroid plexus: 1.2+/-0.5; cerebral blood vessels: 0.6+/-0.1). These results suggest that, even when systemically administered, r-Hu-EPO may have therapeutic potential for stroke via the proliferation of oligodendroglial and endothelial progenitor cells.

Sangue de cordão umbilical para uso autólogo ou grupo de pacientes especiais: [revisão] / The potential therapeutic use of cord blood in autologous transplants or in special patients: a review and update

O sangue de cordão umbilical e placentário (SCUP) é uma rica fonte de células-tronco (CT) hematopoéticas e é amplamente utilizado como substituto da medula óssea em casos de transplante. As células do SCUP possuem vantagens sobre as células da medula óssea (MO), principalmente por serem mais jovens e apresentarem maior taxa proliferativa. Além dos progenitores hematopoéticos, o sangue de cordão umbilical contém progenitores endoteliais e mesenquimais, sugerindo sua possível aplicação nos novos protocolos de terapia celular para diferentes tecidos. Na presente revisão, discutimos a importância do armazenamento do sangue de cordão umbilical autólogo e as pesquisas desenvolvidas para a sua aplicação em doenças degenerativas.

Umbilical Cord Blood is a rich source of hematopoietic stem cells widely used as a substitute of bone marrow (BM) in transplants. Cells from umbilical cord blood present advantages over BM cells, mainly as they are younger and a have higher proliferative rate. Besides hematopoietic stem cells, umbilical cord blood contains endothelial and mesenchymal progenitor cells, suggesting their possible application in cell therapy protocols for different tissues. In this paper, we discuss the importance of autologous umbilical cord blood storage and the research on stem cell transplantation for degenerative diseases.

Sorting and analysis of hematopoietic stem cells and myeloid lineage-committed progenitors using flow cytometry / 中华检验医学杂志

Objective To study the experimental protocol for purification and analysis of hematopoietic stem cells(HSC)and myeloid lineage-committed progenitors.Methods According to differentiation antigen expression pattern on hematopoietic stem cells(HSC) and progenitors during hematopoietic development,HSC and progenitors from bone marrow of 14 healthy mice were analyzed and sorted by magnetic nanoparticles and 4-color or 6-color flow cytometry using multiple antibody panels.Sorted HSC and progenitors were further tested by methylcellulose colony forming unit(CFU)and serial replatingassays.Results The expression of hematopoietic progenitor cells(HPC)was 10-fold higher expression than that of HSC.However,replating activity of common myeloid rogenitors(CMP)was only half of that of HSC.And there was almost 120 replating activity observed in granulocyte/macrophage lineage-restricted progenitors(GMP)and megakaryocyte/erythroeyte lineage-restricted progenitors(MEP).Conclusion Multiparametric flow cytometry could be used to isolate and count HSC and myeloid lineage-committed progenitors accurately.

Culture,purification and differentiation in vitro of human retinal progenitors / 基础医学与临床

Objective To study the expansion and purification of human retinal progenitors cultured in vitro.(Methods Human) embryonic retinal progenitor cells(RPCs) were cultured.The characteristics of neural stem cell were observed.The expression of Nestin was examined flow cytometry(FCM) for the comparsion between the primary and secondary passages.The differentiation of RPCs had been observed after that they had been cultured in different conditions.Results Human RPCs had proliferated and formed cell clusters with a shape of neurosphere.After digestion,the scattered progenitors formed secondary neurosphere-like cell clusters.The cells in those clusters expressed Nestin and were negative for the mature retinal cell proteins.After spontaneous differentiation,the retinal progenitors expressed special proteins of mature retinal cells,including neuron specific enolase(NSE),glial fibrillary acidic protein(GFAP),protein kinase C(PKC) and Syntaxin-1.The expression of Nestin in the secondary culture passage was(39.3?18.9)%,which was significantly higher than that of the primary passage [(28.7?17.7)%](P

Proliferation and Differentiation of Erythroid Progenitors from Cord Blood CD34 (+) Cells Using Extracellular Matrix and Stromal Cells / 대한소아혈액종양학회지

PURPOSE: This study was performed to induce proliferation and differentiation of erythroid progenitors from cord blood CD34 cells using extracellular matrix (ECM) and stromal cells. METHODS: Cord blood mononuclear cells were separated using Ficoll-Hypaque and CD34 cells were purified by Mini-MACS column. Cells were cultured in IMDM medium including 10% FBS and several cytokines (EPO, flt3-ligand, SCF and TPO) upto 14 days. ECM like laminin, collagen IV, fibrinogen and fibronectin were used alone or in combination. Stromal cells were derived from BM mononuclear cells for 4 weeks of culture and irradiated before use. On day 14 of stromal coculture of cord blood CD34 cells, flow cytometric analysis were done with fluorescence isothiocyanate-conjugated (FITC) antihuman glycophorin A antibody for erythroid cells. RESULTS: ECM-treated group showed 16.3~31.3 fold increase of the cell numbers on day 14 and there were no difference from cytokines-treated group. Stromal cells induced great amount of fold increase compared with ECM-treated group on day 7 (25.4 fold vs. 2.2~5.4 fold). The cell numbers increased upto 16.4 fold on day 7, 92.8 fold on day 10, and 198.4 fold on day 14 with stromal cells. Erythroid progenitors expressing glycophorin A increased from 2.78% on day 0 to 21.57% on day 7 and 43.87% on day 14. CONCLUSION: Stromal coculture of cord blood CD34 cells induced marked proliferation and differentiation of erythroid cells compared with cytokines or ECM-treated group. Efficient in vitro erythroid culture might have implications for gene therapy in RBC defects or developing blood substitutes for transfusions.

Synergistic activation of p70S6 kinase associated with stem cell factor in MO7e cells

Stem cell factor (SCF) is an early-acting cytokine inducing proliferative synergy with other cytokines in hematopoietic cells. We earlier showed that p21 was synergistically induced in SCF synergy and the p44/42 MAPK pathway was essential for the transcriptional control of p21. SCF synergy accompanies protein synthesis. p70S6K implicated in translational control in many other systems has not been shown in SCF synergy induced system. GM-CSF dependent human cell line MO7e was stimulated with GM-CSF with SCF, and investigated activation of p70S6K by using phospho-specific antibody. A possible contribution of p70S6K to SCF synergy was examined by measuring p21 induction as a model system. p70S6K was slightly activated by GM-CSF alone and markedly activated by SCF alone. Combined stimulation with these two cytokines synergistically activated p70S6K resulting in persistent activation. Addition of the pathway- specific inhibitors for PI3K or FRAP/TOR, two upstream pathways of p70S6K resulted in abolishment of p70S6K phosphorylation and also significant reduction of p21 protein level. These data suggest that synergistically activated p70S6K by GM-CSF plus SCF involves, at least in part, protein translational control including regulation of p21 protein.

Transplantation of neural stem cells: cellular & gene therapy for hypoxic-ischemic brain injury

We have tracked the response of host and transplanted neural progenitors or stem cells to hypoxic-ischemic (HI) brain injury, and explored the therapeutic potential of neural stem cells (NSCs) injected into mice brains subjected to focal HI injury. Such cells may integrace appropriately into the degenerating central nervous system (CNS), and showed robust engraftment and foreign gene expression within the region of HI inury. They appeared to have migrated preferentially to the site of ischemia, experienced limited proliferation, and differentiated into neural cells lost to injury, trying to repopulate the damaged brain area. The transplantation of exogenous NSCs may, in fact, augment a natural self-repair process in which the damaged CNS "attempts" to mobilize its own pool of stem cells. Providing additional NSCs and trophic factors may optimize this response. Therefore, NSCs may provide a novel approach to reconstituting brains damaged by HI brain injury. Preliminary data in animal models of stroke lends support to these hypotheses.

Quantitative Analysis of Cultured Erythroid Progenitors in Liquid System / 대한혈액학회지

BACKGROUND: Hemopoiesis and erythropoiesis have been studied mainly in immortalized cell lines and semisolid medium. But cell lines do not represent normal erythropoiesis, besides, in semisolid medium the cells are immobilized that it is difficult to do additional immunologic, biochemical, and molecular biologic experiments. In the present study we used a two-phase liquid culture system to isolate and quantify erythroid progenitors from peripheral blood and cord blood. METHODS: Peripheral and cord blood were obtained from three healthy donors and three full-term deliveries, respectively. Mononuclear cells were separated by density gradient centrifugation and were cultured in the first phase at a density of 5x106/mL in alpha- minimal essential medium ( -MEM). After 5~7 days of incubation at 37degrees C in an atmosphere of 5% CO2 with extra humidity, the nonadherent cells were harvested and recultured in the original volume of -MEM containing 10ng/mL stem cell factor and 1U/mL erythropoietin (EPO). Cellular morphology was observed by preparing cytocentrifuge slides stained with Wright Giemsa. On days 8, 10, 12, and 16 of the second phase, hemoglobin (Hb)- containing cells were counted on hemocytometer after staining with acid benzidine and glycophorin A-positive erythroid cells were scored by a flow cytometer. RESULTS: Pronormoblasts first started to appear on days 4~5 in the secondary culture. On day 10 basophilic normoblasts could be seen and on days 12~14 orthochromatic normoblasts were present. Both from peripheral and cord blood the maximum number of benzidine and glycophorin A-positive cells were achieved after 10 days and the total erythroid cell yield was approximately 1x106/mL. CONCLUSION: Using two-phase liquid culture, erythroid cell yield reached 1x106/mL both from peripheral and cord blood. In addition, this culture system permits the study of the effect of various culture conditions and components without terminating the culture, therefore it might provide us a useful experimental tool for studying pathogenesis and therapeutic modalities in genetic erythroid disorders as well as erythroid cell development.

THE PROLIFERATION AND DIFFERENTIATION OF NEURAL PROGENITORS IN THE SN OF PARKINSONIAN RATS / 解剖学报

Objective To observe the proliferation and differentiation of the neural progenitors in the midbrain substantia nigra(SN) of adult rat when dopaminergic neurons(DA neurons) were destroyed. Methods The Parkinson disease model(PD model) was induced in adult SD rats by steriotaxic injection of 6-OHDA into the right striatum,and then assessed by behavioral analysis to screen the qualified models.After varied survival period,the brain of model rats were perfused and fixed by 4% paraformaldehyde,moved the segment of SN,embedded with paraffin and coronally sectioned continuously.The microsections were processed by immunohistochemistry labeling separately the neural progenitors with anti-nestin monoclonal antibody,the dividing cells with anti-PCNA(proliferation cell nuclear antigen),the neuronal precursors with anti-Tuj1(?-tubulin isotype Ⅲ),and DA neurons with anti-TH(tyrosine hydroxylase).The labeled cells were counted under microscope and analyzed statistically.(Results It) was found in the right SN of PD model rats that: 1.Nestin positive(Nestin~+) cells appeared 10d after 6-OHDA injection,became abundant on 14d,declined in number on 17d,and disappeared on 21d.2.Weakly positive PCNA(PCNA~+) cells appeared on 7d.PCNA~+ cells were abundant on 14d,decreased in number from 21d,with only a few positive cells noticed on 28d.3.Tuj1 positive cells appeared in small number on 10d,became abundant on 14d,decreased in number from 17d and dropped nearly to zero on 21d.4.The number of TH positive neurons was significantly less than the normal control(by 24%) on 7d,and became even less as time elapsed.Conclusion When 6-OHDA is injected into the striatum of adult rats to cause degeneration and death of the DA neurons there,there would be a certain period of time in which a number of neural progenitors will be induced to proliferate actively and differentiate toward neuronal cells(except DA neurons).

IN VITRO EFFECTS OF GLIA MATURATION FACTOR ON THE DEVELOPMENT AND DIFFERENTIATION OF CENTRAL MACROGLIAL CELLS / 解剖学报

Optic nerves were dissected from 7-day-old Sprague-Dawley rats, dissociated with collagenase and trypsin, and cultured on poly-L-lysine coated glass coverslips. Cultures were grown in the B-S medium (Bottenstein and Sato, 1979) containing 0.5% fetal calf serum(FCS). All of the coverslip cultures were divided into 4 groups: i. e. Ⅰ, "Ⅰ+GMF", Ⅱ, and "Ⅱ+GMF". Optic nerve glial cells were cocultured with glioblast monolayers in both group Ⅱ and group "Ⅱ+GMF". Glia maturation factor (GMF) was added to the medium in a concentration of 250 ng/ml for group "Ⅰ+GMF" and group "Ⅱ+GMF". No GMF was added to the medium in both group Ⅰ and group Ⅱ. The coverslip cultures were treated with indirect immunofluorescence to identify the cell types of optic nerve. Cells were double-labled with monoclonal gotibody A2B5 and anti-galactocerebroside(GC) monoclonal antibody, or A2B5 and anti-glisl fibrillary acidicprotein (GFAP) antibody. The bipotential glial progenitor cells (A2B5~+, GC~-; or A2B5~+, GFAP~-), mature oligodendrocytes (A2B5~-, GC~+), immature oligodendrocytes(A2B5~+, GC~+), type 1 astrocytes (A2BS~-, GFAP~+) and type 2 astrocytes (A2B5~+, CFAP~+) can be distinguished.After 5 days in culture, the number of cells in group "Ⅱ+GMF" showed a significant increase (P