Achievements and challenges in bioartificial kidney development

Bioartificial kidneys (BAKs) combine a conventional hemofilter in series with a bioreactor unit containing renal epithelial cells. The epithelial cells derived from the renal tubule should provide transport, metabolic, endocrinologic and immunomodulatory functions. Currently, primary human renal proximal tubule cells are most relevant for clinical applications. However, the use of human primary cells is associated with many obstacles, and the development of alternatives and an unlimited cell source is one of the most urgent challenges. BAKs have been applied in Phase I/II and Phase II clinical trials for the treatment of critically ill patients with acute renal failure. Significant effects on cytokine concentrations and long-term survival were observed. A subsequent Phase IIb clinical trial was discontinued after an interim analysis, and these results showed that further intense research on BAK-based therapies for acute renal failure was required. Development of BAK-based therapies for the treatment of patients suffering from end-stage renal disease is even more challenging, and related problems and research approaches are discussed herein, along with the development of mobile, portable, wearable and implantable devices

Differentiation of neurons from neural precursors generated in floating spheres from embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Neural differentiation of embryonic stem (ES) cells is usually achieved by induction of ectoderm in embryoid bodies followed by the enrichment of neuronal progenitors using a variety of factors. Obtaining reproducible percentages of neural cells is difficult and the methods are time consuming.</p> <p>Results</p> <p>Neural progenitors were produced from murine ES cells by a combination of nonadherent conditions and serum starvation. Conversion to neural progenitors was accompanied by downregulation of <it>Oct4 </it>and <it>NANOG </it>and increased expression of <it>nestin</it>. ES cells containing a GFP gene under the control of the <it>Sox1 </it>regulatory regions became fluorescent upon differentiation to neural progenitors, and ES cells with a tau-GFP fusion protein became fluorescent upon further differentiation to neurons. Neurons produced from these cells upregulated mature neuronal markers, or differentiated to glial and oligodendrocyte fates. The neurons gave rise to action potentials that could be recorded after application of fixed currents.</p> <p>Conclusion</p> <p>Neural progenitors were produced from murine ES cells by a novel method that induced neuroectoderm cells by a combination of nonadherent conditions and serum starvation, in contrast to the embryoid body method in which neuroectoderm cells must be selected after formation of all three germ layers.</p

Niche-Independent Symmetrical Self-Renewal of a Mammalian Tissue Stem Cell

Pluripotent mouse embryonic stem (ES) cells multiply in simple monoculture by symmetrical divisions. In vivo, however, stem cells are generally thought to depend on specialised cellular microenvironments and to undergo predominantly asymmetric divisions. Ex vivo expansion of pure populations of tissue stem cells has proven elusive. Neural progenitor cells are propagated in combination with differentiating progeny in floating clusters called neurospheres. The proportion of stem cells in neurospheres is low, however, and they cannot be directly observed or interrogated. Here we demonstrate that the complex neurosphere environment is dispensable for stem cell maintenance, and that the combination of fibroblast growth factor 2 (FGF-2) and epidermal growth factor (EGF) is sufficient for derivation and continuous expansion by symmetrical division of pure cultures of neural stem (NS) cells. NS cells were derived first from mouse ES cells. Neural lineage induction was followed by growth factor addition in basal culture media. In the presence of only EGF and FGF-2, resulting NS cells proliferate continuously, are diploid, and clonogenic. After prolonged expansion, they remain able to differentiate efficiently into neurons and astrocytes in vitro and upon transplantation into the adult brain. Colonies generated from single NS cells all produce neurons upon growth factor withdrawal. NS cells uniformly express morphological, cell biological, and molecular features of radial glia, developmental precursors of neurons and glia. Consistent with this profile, adherent NS cell lines can readily be established from foetal mouse brain. Similar NS cells can be generated from human ES cells and human foetal brain. The extrinsic factors EGF plus FGF-2 are sufficient to sustain pure symmetrical self-renewing divisions of NS cells. The resultant cultures constitute the first known example of tissue-specific stem cells that can be propagated without accompanying differentiation. These homogenous cultures will enable delineation of molecular mechanisms that define a tissue-specific stem cell and allow direct comparison with pluripotent ES cells

Synaptically-Competent Neurons Derived from Canine Embryonic Stem Cells by Lineage Selection with EGF and Noggin

Pluripotent stem cell lines have been generated in several domestic animal species; however, these lines traditionally show poor self-renewal and differentiation. Using canine embryonic stem cell (cESC) lines previously shown to have sufficient self-renewal capacity and potency, we generated and compared canine neural stem cell (cNSC) lines derived by lineage selection with epidermal growth factor (EGF) or Noggin along the neural default differentiation pathway, or by directed differentiation with retinoic acid (RA)-induced floating sphere assay. Lineage selection produced large populations of SOX2+ neural stem/progenitor cell populations and neuronal derivatives while directed differentiation produced few and improper neuronal derivatives. Primary canine neural lines were generated from fetal tissue and used as a positive control for differentiation and electrophysiology. Differentiation of EGF- and Noggin-directed cNSC lines in N2B27 with low-dose growth factors (BDNF/NT-3 or PDGFαα) produced phenotypes equivalent to primary canine neural cells including 3CB2+ radial progenitors, MOSP+ glia restricted precursors, VIM+/GFAP+ astrocytes, and TUBB3+/MAP2+/NFH+/SYN+ neurons. Conversely, induction with RA and neuronal differentiation produced inadequate putative neurons for further study, even though appropriate neuronal gene expression profiles were observed by RT-PCR (including Nestin, TUBB3, PSD95, STX1A, SYNPR, MAP2). Co-culture of cESC-derived neurons with primary canine fetal cells on canine astrocytes was used to test functional maturity of putative neurons. Canine ESC-derived neurons received functional GABAA- and AMPA-receptor mediated synaptic input, but only when co-cultured with primary neurons. This study presents established neural stem/progenitor cell populations and functional neural derivatives in the dog, providing the proof-of-concept required to translate stem cell transplantation strategies into a clinically relevant animal model

Pleiotropic effects of heading and marker genes on variation

以具有台中65號遺傳背景之抽穗及標識基因之近同源系為材料, 探 討此等基因對農藝性狀株內變異性之影響,結果簡要如下:1.抽穗基因對 農藝性狀株內變異性有多效作用,其程度因基因及性狀之不同而異,Ef-1 b對分蘗長度及稔實率Ef-1r對分蘗長度,穗長,不稔粒數及稔實率CV有增 大作用.ef-3對稔實率,ef-2不稔粒數CV值有增大作用.ef-4 則對調查性 狀CV值無顯著影響.2.標識基因對農藝性狀株內變異性有多效作用,其程 度因基因及性狀之不同而異,A對分蘗長度,百粒重;Rc對百粒重;wx 對分 蘗長度,穗重及稔實粒重,Dn-1對一次枝梗數,稔實率CV值有增大作用;g- 1基因對所有調查性狀(除百粒重及稔實率外)CV值有增大作用;An-4,fs- 1則對所有調查性狀CV值皆無顯著影響

以微滴定盤三明治式DNA雜交法及聚合�t鏈鎖反應檢測食品中之沙門氏菌

依微滴定盤三明治式DNA雜交法的研究結果，使用紫外線照射確實有助於捕捉探 針固定於微滴定盤孔洞上。而以由1.8 Kb DNA經EcoR Ⅱ 切割所得之不重疊之0.5 ▔▔ kb和1.3 kb DNA分別充當捕捉探針及偵測探針，進行沙門氏菌的檢測專一性探討裡 ，九株不同血清型的沙門氏菌之OD410 值均高於0.4 ，而三種非沙門氏菌，包括 E.coli，Citrobacter 及Shigella均低於0.2 ，因此，應用此系統於沙門氏菌之檢 ▔ ▔▔ ▔▔▔▔▔▔ ▔▔▔▔ 測，具有相當高的專一性。而檢測靈敏度的研究結果顯示，純菌只要10cells/ml 即可檢出，而食品中之沙門氏菌則需１０6 - １０cells/g foods 才可檢出。 以PCR檢測沙門氏菌之實驗結果，TS11/TS5, TS11/TS4兩組引子均具有檢測專一 性，用TS11/TS4當作PCR 引子進行純培養及食品（包括雞肉，豬肉，牛肉，鴨肉， cheese）中沙門氏菌之檢測時，其靈敏度均高達10°cells/ml或g食品，而進一步 控制短時間增殖培養及稀釋倍數，以PCR 可區分沙門氏菌之10cells/ml非生菌與 10°cells/ml生菌。為了縮短聚合鏈鎖反應的時間，以TS11/TS5這組引子在94℃ /1.5 min, 68℃/2.0 min的條件下進行二階段PCR ，其專一性甚佳，五株當做檢測 菌株之不同血清型的沙門氏菌均有375 bp的PCR 產物，而五株Citrobacter 與其他 五株非沙門氏菌則無此PCR 產物。 ▔▔▔▔▔