Potassium channels play a critical role in neurogenesis and establishing electrical excitability of neurons in model organisms. Much less is known about the involvement of potassium channels in human development. We have investigated the potential role of potassium channels in neurogenesis of stem cells isolated from human umbilical cord matrix. We hypothesize that potassium channels expressed in stem cells play an important role in both development and repair in the brain. Here we demonstrate expression of a number of potassium channels during the course of neuronal differentiation of stem cells derived from human umbilical cord matrix based on immunodetection and whole cell patch clamp analysis of potassium currents. We explored the effects of blocking potassium channels that were identified on neuronal differentiation. Our findings show that blocking different potassium channels had varying effects with blocking the large conductance calcium activated potassium channel being most profound. Neuronal differentiation was inhibited based on the lack of morphological changes into neuronal phenotypes and the altered expression of a number of genes associated with neuronal differentiation. These results suggest that potassium channels play a critical role in differentiation of stem cells into neuronal phenotypes. Funded by NIH Grant Number P20 RR016475, NIH (R01NS036124) and the State of Kansas.

Stem cells are being evaluated in numerous human clinical trials and are commercially used in veterinary medicine to treat horses and dogs. Stem cell differentiation, homing to disease sites, growth and cytokine factor modulation, and low antigenicity contribute to their therapeutic success. Bone marrow and adipose tissue are the two most common sources of adult-derived stem cells in animals. We report on the existence of an alternative source of primitive, multipotent stem cells from the equine umbilical cord cellular matrix (Wharton's jelly). Equine umbilical cord matrix (EUCM) cells can be cultured, cryogenically preserved, and differentiated into osteo-, adipo-, chondrogenic, and neuronal cell lineages. These results identify a source of stem cells that can be non-invasively collected at birth and stored for future use in that horse or used as donor cells for treating unrelated horses.

Stem cells from the human umbilical cord matrix (HUCM) cells express many proteins important for regulation of embryonic and adult stem cells and can be differentiated into cells from mesodermal and ectodermal lineages. Here we describe differentiation of HUCM stem cells into hepatocytes, an endodermal cell‐type. HUCM‐derived hepatocytes stored glycogen, indocyanine green and took up low density lipoprotein while the undifferentiated HUCM cells did not. HUCM‐derived hepatocytes expressed hepatocyte markers: albumin, HNF3‐beta, HNF4‐alpha, and glutamine synthetase, which are not expressed by undifferentiated HUCM cells. Smooth muscle actin and nanog, proteins normally expressed in undifferentiated HUCM cells, were no longer expressed. Transcription factors and P450's that are associated with liver metabolism were expressed in the HUCM‐derived hepatocytes and further induced by treatment with drugs. Changes in gene expression determined by real‐time PCR showed that stem cell gene expression is decreased in HUMC‐derived hepatocytes while .expression of genes associated with metabolic pathways in the liver were increased. These findings suggest that this abundant source of stem cells could be used for drug development in addition to cell‐based therapy for liver disease. Funded by NIH P20 RR016475 and the State of Kansas.

ES‐like stem cells from the human umbilical cord matrix (HUCM) express the ES transcription factors Oct‐4 and nanog. HUCM cells also express adult stem cell markers so appear to be intermediate between ES and tissue‐specific adult stem cells. HUCM cells can be differentiated into endodermal, mesodermal and ectodermal lineages making them candidates for cell‐based therapies. We explored changes in gene expression after differentiation of HUCM cells into different cell lineages. In particular, we determined changes in expression of genes associated with undifferentiated embryonic stems (Oct4 and nanog); adult stem cells (CD146, CD90 and smooth muscle actin) and immune complexes (HLA‐ABC and MHC II). We found that Oct4 and nanog expression was decreased to non‐detectable levels after differentiation of HUCM cells and that smooth muscle actin was initially greatly diminished, becoming disorganized and eventually below the level of detection. HLA‐ABC and MHC‐II are not detectable in undifferentiated HUCM cells or in some cases HLA‐ABC expression was very weak. Expression of MHC‐II remained below the level of detection and HLA‐ABC remained very low after in vitro differentiation. This work will establish whether HUCM cells are potential candidates for allogenic cell‐based therapies. Funded by NIH Grant Number P20 RR016475, NIH (R01NS036124) and the State of Kansas.

The temporomandibular joint (TMJ) presents many problems in modern musculoskeletal medicine. Patients who suffer from TMJ disorders often experience a major loss in quality of life due to the debilitating effects that TMJ disorders can have on everyday activities. Cartilage tissue engineering can lead to replacement tissues that could be used to treat TMJ disorders. In this study, a spinner flask was used for a period of 6 days to seed polyglycolic acid (PGA) scaffolds with either TMJ condylar chondrocytes or mesenchymal-like stem cells derived from human umbilical cord matrix (HUCM). Samples were then statically cultured for 4 weeks either in growth medium containing chondrogenic factors or in control medium. Immunohistochemical staining of HUCM constructs after 4 weeks revealed a strong presence of collagen I and minute amounts of collagen II, whereas TMJ constructs revealed little collagen I and no collagen II. The HUCM constructs were shown to contain more GAGs than the TMJ constructs quantitatively at week 0 and histologically at week 4. Moreover, the cellularity of HUCM constructs was 55% higher at week 0 and nearly twice as high after 4 weeks, despite being seeded at the same density. The increased level of biosynthesis and higher cellularity of HUCM constructs clearly demonstrates that the HUCM stem cells outperformed the TMJ condylar cartilage cells under the prescribed conditions. HUCM stem cells may therefore be an attractive alternative to condylar cartilage cells for TMJ tissue engineering applications. Further, given the availability and ease of obtaining HUCM stem cells, these findings may have far-reaching implications, leading to novel developments in both craniofacial and orthopaedic tissue replacement therapies.

PURPOSE:Organ transplant patients treated with cyclosporine-A (CsA) often exhibit weight loss and muscle weakness. The cellular target of CsA, calcineurin, has been implicated in maintenance of muscle fiber size and in expression of the type I skeletal muscle phenotype. We hypothesized that CsA treatment would cause fiber atrophy, as well as increase type IIa myosin heavy chain (MHC) content and oxidative enzyme activities in the soleus muscle. METHODS:Rats were treated with CsA for 21 d (20 mg.kg(-1).d(-1); N = 16) and compared with control rats given olive oil vehicle (Veh; N = 16). Soleus muscles were excised bilaterally. MHC content was determined by gel electrophoresis, oxidative enzyme activities by spectrophotometric methods, and fiber type and size by histochemistry. RESULTS:Lymphocyte count was depressed in CsA rats (P < 0.05), indicating treatment efficacy. Type IIa MHC content was increased in the soleus muscle with CsA (Veh, 10.4 +/- 1.7%; CsA, 15.1 +/- 2.0; P < 0.05) at the expense of type I MHC. Soleus muscle oxidative enzyme activities were also increased with CsA treatment (P < 0.05). Soleus muscle atrophy occurred, reflected by a 22% decrease in fiber cross-sectional area (Veh, 3255 +/- 105 microm(2); CsA, 2533 +/- 125; P < 0.05). CONCLUSION:These findings indicate that CsA treatment is associated with changes in skeletal muscle fiber size and phenotype. The former may underlie clinical symptoms of transplant patients treated with CsA.

Potential therapeutic effects of Oct-4-positive rat umbilical cord matrix (RUCM) cells in treating cerebral global ischemia were evaluated using a reproducible model of cardiac arrest (CA) and resuscitation in rats. Animals were randomly assigned to four groups: A, sham-operated; B, 8-minute CA without pretreatment; C, 8-minute CA pretreated with defined media; and D, 8-minute CA pretreated with Oct-4(+) RUCM cells. Pretreatment was done 3 days before CA by 2.5-mu l microinjection of defined media or approximately 104 Oct-4(+) RUCM cells in left thalamic nucleus, hippocampus, corpus callosum, and cortex. Damage was assessed histologically 7 days after CA and was quantified by the percentage of injured neurons in hippocampal CA1 regions. Little damage (approximately 3%-4%) was found in the sham group, whereas 50%-68% CA1 pyramidal neurons were injured in groups B and C. Pretreatment with Oct-4(+) RUCM cells significantly (p < .001) reduced neuronal loss to 25%-32%. Although the transplanted cells were found to have survived in the brain with significant migration, few were found directly in CA1. Therefore, transdifferentiation and fusion with host cells cannot be the predominant mechanisms for the observed protection. The Oct-4(+) RUCM cells might repair nonfocal tissue damage by an extracellular signaling mechanism. Treating cerebral global ischemia with umbilical cord matrix cells seems promising and worthy of further investigation.

Restoration of chloride conductance via introduction of an anion-selective pore, formed by a channel-forming peptide, has been hypothesized as a novel treatment modality for patients with cystic fibrosis. Delivery of these peptides from an aqueous environment in the absence of organic solvents is paramount. M2GlyR peptides, designed based on the glycine receptor, insert into lipid bilayers and polarized epithelial cells and assemble spontaneously into chloride-conducting pores. Addition of 4 lysine residues to either terminus increases the solubility of M2GlyR peptides. Both orientations of the helix within the membrane form an anion-selective pore, however, differences in solubility, associations and channel-forming activity are observed. To determine how the positioning of the lysine residues affects these properties, structural characteristics of the lysyl-modified peptides were explored utilizing chemical cross-linking, NMR and molecular modeling. Initial model structures of the a-helical peptides predict that lysine residues at the COOH-terminus form a capping structure by folding back to form hydrogen bonds with backbone carbonyl groups and hydroxyl side chains of residues in the helical segment of the peptide. In contrast, lysine residues at the NH2-terminus form fewer H-bonds and extend away from the helical backbone. Results from NMR and chemical cross-linking support the model structures. The C-cap formed by H-bonding of lysine residues is likely to account for the different biophysical properties observed between NH2- and COOH-terminal-modified M2GlyR peptides.

ABSTRACT Bovine immunodeficiency virus (BIV) and Jembrana disease virus (JDV) are closely related bovine lentiviruses that are difficult to distinguish by presently available diagnostic methods. Recently, in our laboratory, a monoclonal antibody (MAb; MAb 10H1) against the BIV Gag protein identified a differential epitope, located at the 6.4-kDa N terminus of a 29-kDa Gag capsid protein, which was absent in JDV. To define the essential amino acids of the epitope, a series of primers within the 163 bp of DNA corresponding to the 6.4-kDa protein were designed. The full-length 163-bp DNA fragment and the smaller DNA fragments with deletions were amplified by PCR and then cloned into pQE32 vectors for protein expression studies. The expressed proteins were analyzed with MAb 10H1 by Western blotting. The differential epitope has been narrowed to a 26-amino-acid region (R121 to R146), which includes 6 residues of p16 MA (where MA represents the matrix protein) and 20 residues of p2L. A synthetic peptide corresponding to the putative 26-amino-acid epitope blocked MAb 10H1 binding to the expressed peptide. These experiments revealed that the epitope spans the cleavage site between p16 MA and p2L and presumably will be valuable in distinguishing the two viruses.

Restoration of chloride conductance via the introduction of an anion selective pore, formed by a channel-forming peptide, has been hypothesized as a novel treatment modality for patients with cystic fibrosis (CF). Delivery of these peptide sequences to airway cells from an aqueous environment in the absence of organic solvents is paramount. New highly soluble COOH- and NH2-terininal truncated peptides, derived from the second transmembrane segment of the glycine receptor alpha-subunit (M2GlyR), were generated, with decreasing numbers of amino acid residues. NH2-terminal lysyl-adducted truncated peptides with lengths of 22, 25, and 27 amino acid residues are equally able to stimulate short circuit current (I-SC). Peptides with as few as 16 amino acid residues are able to stimulate I-SC, although to a lesser degree. In contrast, COOH-terminal truncated peptides show greatly reduced induced I-SC values for all peptides fewer than 27 residues in length and show no measurable activity for peptides fewer than 21 residues in length. CD spectra for both the NH2- and COOH-truncated peptides have random structure in aqueous solution, and those sequences that stimulated the highest maximal I-SC are predominantly helical in 40% trifluoroethanol. Peptides with a decreased propensity to form helical structures in TFE also failed to stimulate I-SC. Palindromic peptide sequences based on both the NH2- and COOH-terminal halves of M2GlyR were synthesized to test roles of the COOH- and NH2-terminal halves of the molecule in solution aggregation and channel forming ability. On the basis of the study presented here, there are distinct, nonoverlapping regions of the M2GlyR sequence that define solution aggregation and membrane channel assembly. Peptides that eliminate solution aggregation with complete retention of channel forming activity were generated.