Prospective purification of perivascular presumptive mesenchymal stem cells from human adipose tissue: process optimization and cell population metrics across a large cohort of diverse demographics.

BACKGROUND: Adipose tissue is an attractive source of mesenchymal stem cells (MSC) as it is largely dispensable and readily accessible through minimally invasive procedures such as liposuction. Until recently MSC could only be isolated in a process involving ex-vivo culture and their in-vivo identity, location and frequency remained elusive. We have documented that pericytes (CD45-, CD146+, and CD34-) and adventitial cells (CD45-, CD146-, CD34+) (collectively termed perivascular stem cells or PSC) represent native ancestors of the MSC, and can be prospectively purified using fluorescence activated cell sorting (FACS). In this study we describe an optimized protocol that aims to deliver pure, viable and consistent yields of PSC from adipose tissue. We analysed the frequency of PSC within adipose tissue, and the effect of patient and procedure based variables on this yield. METHODS: Within this twin centre study we analysed the adipose tissue of n = 131 donors using flow cytometry to determine the frequency of PSC and correlate this with demographic and processing data such as age, sex, BMI and cold storage time of the tissue. RESULTS: The mean number of stromal vascular fraction (SVF) cells from 100 ml of lipoaspirate was 34.4 million. Within the SVF, mean cell viability was 83 %, with 31.6 % of cells being haematopoietic (CD45+). Adventitial cells and pericytes represented 33.0 % and 8 % of SVF cells respectively. Therefore, a 200 ml lipoaspirate would theoretically yield 23.2 million viable prospectively purified PSC - sufficient for many reconstructive and regenerative applications. Minimal changes were observed in respect to age, sex and BMI suggesting universal potential application. CONCLUSIONS: Adipose tissue contains two anatomically and phenotypically discreet populations of MSC precursors - adventitial cells and pericytes - together referred to as perivascular stem cells (PSC). More than 9 million PSC per 100 ml of lipoaspirate can be rapidly purified to homogeneity using flow cytometry in clinically relevant numbers potentially circumventing the need for purification and expansion by culture prior to clinical use. The number and viability of PSC are minimally affected by patient age, sex, BMI or the storage time of the tissue, but the quality and consistency of yield can be significantly influenced by procedure based variables.

The ShcA SH2 domain engages a 14-3-3/PI3'K signaling complex and promotes breast cancer cell survival.

The ShcA adapter protein transmits activating signals downstream of receptor and cytoplasmic tyrosine kinases through the establishment of phosphotyrosine-dependent complexes. In this regard, ShcA possesses both a phosphotyrosine-binding domain (PTB) and Src homology 2 domain (SH2), which bind phosphotyrosine residues in a sequence-specific manner. Although the majority of receptor tyrosine kinases expressed in breast cancer cells bind the PTB domain, very little is known regarding the biological importance of SH2-driven ShcA signaling during mammary tumorigenesis. To address this, we employed transgenic mice expressing a mutant ShcA allele harboring a non-functional SH2 domain (ShcR397K) under the transcriptional control of the endogenous ShcA promoter. Using transplantation approaches, we demonstrate that SH2-dependent ShcA signaling within the mammary epithelial compartment is essential for breast tumor outgrowth, survival and the development of lung metastases. We further show that the ShcA SH2 domain activates the AKT pathway, potentially through a novel SH2-mediated complex between ShcA, 14-3-3ζ and the p85 regulatory subunit of phosphatidylinositol 3 (PI3') kinase. This study is the first to demonstrate that the SH2 domain of ShcA is critical for tumor survival during mammary tumorigenesis.

Mammary gland neoplasia: insights from transgenic mouse models.

Current theories of breast cancer progression have been greatly influenced by the development and refinement of mouse transgenic and gene targeting technologies. Early transgenic mouse models confirmed the involvement of oncogenes, previously implicated in human breast cancer, by establishing a causal relationship between overexpression or activation of these genes and mammary tumorigenesis. More recently, the importance of genes located at sites of loss of heterozygosity in human breast cancer have been examined in mice by their targeted disruption via homologous recombination. The union of these two approaches allows the generation of complex animal models that more accurately reflect the multistep nature of human breast cancer. This review will examine how the study of transgenic mice has increased our understanding of the molecular events responsible for oncogenic transformation of the mammary gland. BioEssays 22:554-563, 2000.

Amplification of the neu/erbB-2 oncogene in a mouse model of mammary tumorigenesis.

The neu (c-erbB-2, Her-2) protooncogene is amplified and overexpressed in 20-30% of human breast cancers. Although transgenic mouse models have illustrated the role of Neu in the induction of mammary tumors, Neu expression in these models is driven by a strong viral promoter of questionable relevance to the human disease. To ascertain whether expression of activated Neu under the control of the endogenous promoter in the mammary gland could induce mammary tumors we have generated mice that conditionally express activated Neu under the transcriptional control of the intact endogenous Neu promoter. Expression of oncogenic neu in the mammary gland resulted in accelerated lobulo-alveolar development and formation of focal mammary tumors after a long latency period. However, expression of activated Neu under the normal transcriptional control of the endogenous promoter was not sufficient for the initiation of mammary carcinogenesis. Strikingly, all mammary tumors bear amplified copies (2-22 copies) of the activated neu allele relative to the wild-type allele and express highly elevated levels of neu transcript and protein. Thus, like human erbB-2-positive breast tumors, mammary tumorigenesis in this mouse model requires the amplification and commensurate elevated expression of the neu gene.

Strain-dependent myeloid hyperplasia, growth deficiency, and accelerated cell cycle in mice lacking the Rb-related p107 gene.

To investigate the function of the Rb-related p107 gene, a null mutation in p107 was introduced into the germ line of mice and bred into a BALB/cJ genetic background. Mice lacking p107 were viable and fertile but displayed impaired growth, reaching about 50% of normal weight by 21 days of age. Mutant mice exhibited a diathetic myeloproliferative disorder characterized by ectopic myeloid hyperplasia in the spleen and liver. Embryonic p107(-/-) fibroblasts and primary myoblasts isolated from adult p107(-/-) mice displayed a striking twofold acceleration in doubling time. However, cell sort analysis indicated that the fraction of cells in G1, S, and G2 was unaltered, suggesting that the different phases of the cell cycle in p107(-/-) cells was uniformly reduced by a factor of 2. Western analysis of cyclin expression in synchronized p107(-/-) fibroblasts revealed that expression of cyclins E and A preceded that of D1. Mutant embryos expressed approximately twice the normal level of Rb, whereas p130 levels were unaltered. Lastly, mutant mice reverted to a wild-type phenotype following a single backcross with C57BL/6J mice, suggesting the existence of modifier genes that have potentially epistatic relationships with p107. Therefore, we conclude that p107 is an important player in negatively regulating the rate of progression of the cell cycle, but in a strain-dependent manner.

Herpes simplex virus inhibits host cell splicing, and regulatory protein ICP27 is required for this effect.

While the majority of metazoan genes and those of the DNA viruses which infect them contain introns which require RNA splicing, herpes simplex virus type 1 (HSV-1) encodes relatively few spliced products. We previously showed that the HSV-1 immediate-early protein ICP27 decreased the levels of spliced target mRNAs in transfections and spliced cellular mRNAs during infection, suggesting that ICP27 may function in impairing host cell splicing. Here, we show that during infections with the wild type, but not in infections with an ICP27 viral mutant termed 27-LacZ, precursor RNA accumulated for a virus transcript which contained introns. Pre-mRNA accumulation in the nucleus was greater than that in the cytoplasm, indicating that splicing rather than transport was affected. Furthermore, splicing of a beta-globin pre-mRNA substrate was inhibited in nuclear extracts from wild-type-infected cells but not in extracts from cells infected with 27-LacZ. The inhibitory activity in extracts from wild-type-infected cells was able to reduce the splicing efficiency of competent extracts in biochemical complementation assays. ICP27 appeared to be responsible for this decrease, because the splicing activity of an extract from cells infected with an ICP27 ts mutant was significantly reduced after incubation of the extract at the permissive temperature to allow renaturation of the conformationally defective ICP27 protein. These results strongly suggest that HSV-1 infection inhibits host cell splicing through the action of ICP27.

Novel activating mutations in the neu proto-oncogene involved in induction of mammary tumors.

Amplification of the Neu/c-erbB-2 receptor tyrosine kinase has been implicated as an important event in the genesis of human breast cancer. Indeed, transgenic mice bearing either an activated form of neu or the wild-type proto-oncogene under the transcriptional control of the mouse mammary tumor virus promoter-enhancer frequently develop mammary carcinomas (L. Bouchard, L. Lamarre, P. J. Tremblay, and P. Jolicoeur, Cell 57:931-936, 1989; C. T. Guy, M. A. Webster, M. Schaller, T. J. Parson, R. D. Cardiff, and W. J. Muller, Proc. Natl. Acad. Sci. USA 89:10578-10582, 1992; W. J. Muller, E. Sinn, R. Wallace, P. K. Pattengale, and P. Leder, Cell 54:105-115, 1988). Induction of mammary tumors in transgenic mice expressing the wild-type Neu receptor is associated with activation of the receptor's intrinsic tyrosine kinase activity (Guy et al., Proc. Natl. Acad. Sci. USA 89:10578-10582, 1992). Here, we demonstrate that activation of Neu in these transgenic mice occurs through somatic mutations located within the transgene itself. Sequence analyses of these mutations revealed that they contain in-frame deletions of 7 to 12 amino acids in the extracellular region proximal to the transmembrane domain. Introduction of these mutations into a wild-type neu cDNA results in an increased transforming ability of the altered Neu tyrosine kinase. These observations suggest that oncogenic activation of Neu in mammary tumorigenesis frequently occurs by somatic mutation.

Phosphorylation of Sindbis virus nsP3 in vivo and in vitro.

nsP3 is one of four viral nonstructural proteins required for RNA replication of Sindbis virus. In this report, post-translational modifications of nsP3 which occur in both vertebrate and mosquito cell cultures have been examined. In pulse-chase experiments, analyzed by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, nsP3 was initially observed as a single species (termed nsP3a, approximately 76 kDa) which was gradually converted to slower mobility forms ranging from 78 kDa (termed nsP3b) to 106 kDa (termed nsP3c). The slower mobility forms, but not nsP3a or the other nonstructural proteins, could be labeled in vivo with [32P]orthophosphate. Treatment of nsP3 immunoprecipitates with calf intestinal alkaline phosphatase converted the slower mobility forms to nsP3a. Phosphoamino acid analysis of nsP3b and nsP3c demonstrated that both contained phosphoserine and phosphothreonine but not phosphotyrosine, nsP34, a polyprotein produced by readthrough of the in-frame opal codon preceding nsP4, was also phosphorylated on serine and threonine residues. nsP3 phosphorylation did not require ongoing RNA synthesis since phosphorylated forms were also observed in the absence of Sindbis-specific RNA synthesis. Furthermore, when immunoprecipitates of nsP3 were incubated with [gamma-32P]ATP in the presence of Mg2+ or Mn2+, a kinase activity which was able to phosphorylate nsP3 on serine and threonine residues in vitro was detected. This kinase activity was inhibited by heparin, was activated by spermidine, and could utilize GTP and ATP as the phosphate donor. These latter properties are similar to those of cellular casein kinase II. Although it is possible that this nsP3-associated kinase is of cellular origin, autophosphorylation of nsP3 has not been excluded.

Cleavage-site preferences of Sindbis virus polyproteins containing the non-structural proteinase. Evidence for temporal regulation of polyprotein processing in vivo.

The non-structural proteins of Sindbis virus, nsP1, 2, 3 and 4, are produced upon cleavage of polyproteins P123 and P1234 by a proteinase residing in nsP2. We used cell free translation of SP6 transcripts to study the proteolytic activity of nsP2 and of nsP2-containing polyproteins. To generate polyprotein enzymes, a set of plasmids was made in which cleavage sites were eliminated and new initiation and termination codons introduced by in vitro mutagenesis. As a substrate, we used a polyprotein in which the nsP2 proteinase had been inactivated by a single amino acid substitution. All nsP2-containing polyproteins cleaved the nsP1/2 site in trans. However, proteinases containing nsP1 were unable to cleave the nsP2/3 site. Furthermore, only proteinases containing nsP3 could cleave the nsP3/4 site. These differences in cleavage site specificity result in a temporal regulation of processing in vivo. At 1.7 h post infection P123 and nsP4 accumulated and only small amounts of P34 were found. However, at 4 h post infection P123 was processed rapidly and P34 was produced rather than nsP4. Since nsP4 is thought to be the viral RNA polymerase, the temporal regulation of the nsP4/P34 ratio may be responsible for the temporal regulation of RNA synthesis.

Synthesis and processing of the nonstructural polyproteins of several temperature-sensitive mutants of Sindbis virus.

We have examined the synthesis and processing of nonstructural polyproteins by several temperature-sensitive mutants of Sindbis virus, representing the four known RNA-minus complementation groups. Four mutants that possess mutations in the C-terminal domain of nonstructural protein nsP2 all demonstrated aberrant processing patterns when cells infected with these mutants were shifted from a permissive (30 degrees) to a nonpermissive (40 degrees) temperature. Mutants ts17, ts18, and ts24 showed severe defects in processing of nonstructural polyproteins at 40 degrees, whereas ts7 showed only a minor defect. In each case, cleavage of the bond between nsP2 and nsP3 was greatly reduced whereas cleavage between nsP1 and nsP2 occurred almost normally, giving rise to a set of polyprotein precursors not seen in wild-type-infected cells at this stage of infection. The nsP1 produced by these mutants was unstable and only small amounts could be detected in infected cells at the nonpermissive temperature. Submolar quantities of nsP2 were also present. We suggest that nsP1 and nsP2 may function as a complex and that free nsP1, and possibly nsP2, is degraded. Cleavage between nsP3 and nsP4 appeared to be normal in the mutants except in the case of ts17, where upon shift to 40 degrees P34 was unstable and nsP4 accumulated. We propose that the change in the P34/nsP4 ratio upon shift is responsible for the previously observed temperature sensitivity of subgenomic 26 S RNA synthesis in ts17 and for the failure of the mutant to regulate minus strand synthesis at 40 degrees. Other mutations tested, including ts21, which is found in the N-terminal half of nsP2, ts11, which has a mutation in nsP1, and ts6, which has a mutation in nsP4, all demonstrated nonstructural polyprotein processing indistinguishable from that in wild-type-infected cells. These results support our conclusion, based upon deletion mapping studies, that the C-terminal domain of nsP2 contains the nonstructural proteinase activity.

Processing the nonstructural polyproteins of sindbis virus: nonstructural proteinase is in the C-terminal half of nsP2 and functions both in cis and in trans.

The processing of the Sindbis virus nonstructural polyprotein translated in vitro has been studied. When Sindbis virus genomic RNA was translated in a reticulocyte lysate, polyprotein P123 was cleaved efficiently to produce nsP1, nsP2, and nsP3. Inhibition of this processing by anti-nsP2 antibodies, but not by antibodies specific for nsP1, nsP3, or nsP4, suggested that the viral proteinase was present in nsP2. To localize the proteolytic activity more precisely, deletions were made in a full-length cDNA clone of Sindbis virus, and RNA was transcribed from these constructs with SP6 RNA polymerase and translated in vitro. Although virtually all of the nsP1, nsP3, and nsP4 sequences could be deleted without affecting processing, deletions in the N-terminal half of nsP2 led to aberrant processing, and deletions in the C-terminal half abolished proteolysis. However, inactive polyproteins containing the nsP2 deletions could be processed by exogenously supplied proteins translated from virion RNA, demonstrating that cleavage was virus specific and not due to a protease present in the reticulocyte lysate and that the deleted polyproteins still served as substrates for the enzyme. From these results and from experiments in which processing was studied at increasingly higher dilution, we have concluded the following: (i) the viral nonstructural proteinase is located in the C-terminal half of nsP2; (ii) in the P123 precursor the cleavage between nsP2 and nsP3 occurs efficiently as a bimolecular reaction (in trans) to remove nsP3, while the bond between nsP1 and nsP2 is cleaved inefficiently, but detectably, in trans, but no autoproteolysis of P123 was detected; (iii) once nsP3 has been removed, the bond between nsP1 and nsP2 in the P12 precursor is cleaved efficiently by autoproteolysis (in cis). This mode of processing leads to a slow rate of cleavage, particularly early in infection, suggesting that the polyproteins might play roles in virus RNA replication distinct from those of the cleaved products. A hypothesis is presented that the proteinase is a thiol protease related to papain.

Processing the nonstructural polyproteins of Sindbis virus: study of the kinetics in vivo by using monospecific antibodies.

Plasmids were constructed which contained a large portion of each of the four nonstructural genes of Sindbis virus fused to the N-terminal two-thirds of the trpE gene of Escherichia coli. The large quantity of fusion protein induced from cells containing these plasmids was subsequently used as an antigen to generate polyclonal antisera in rabbits. Each antiserum was specific for the corresponding nonstructural protein and allowed ready identification of each nonstructural protein and of precursors containing the sequences of two or more nonstructural proteins. These antisera were used to determine the stability of the mature nonstructural proteins and to examine the kinetics of processing of the nonstructural proteins from their respective precursors in vivo. Pulse-chase experiments showed that the precursor P123 is cleaved with a half-life of approximately 19 min to produce P12 and nsP3; P12 is then cleaved with a half-life of approximately 9 min to produce nsP1 and nsP2. Thus, although the rate of cleavage between nsP1 and nsP2 is faster than that between nsP2 and nsP3, the latter cleavage must occur first and is therefore the rate-limiting step. The rate at which P34 is chased suggests that the cleavage between nsP3 and nsP4 is the last to occur; however the regulation of nsP4 function in Sindbis virus-infected cells may be even more complex than was previously thought. The products nsP1 and nsP2 (and nsP4) are relatively stable; nsP3, however, is unstable, with a half-life of about 1 h, and appears to be modified to produce heterodisperse, higher-molecular-mass forms. In general, the processing schemes used by Sindbis virus and Semliki Forest virus appear very similar, the major difference being that most nsP3 in Sindbis virus results from termination at an opal condon, whereas in Semliki Forest virus cleavage of the P34 precursor is required.

Computed tomography scanning after chymopapain injection for herniated nucleus pulposus. A prospective study.

Fifty consecutive patients treated with chymopapain injection for a clinical and radiographic diagnosis of herniated nucleus pulposus were evaluated prospectively. All patients had a prechymopapain computed tomography (CT) scan and a three-month postinjection CT scan. In addition, ten patients (20%) had a six-month postinjection CT scan. All scans were interpreted blindly. Only six patients (12%) had obvious changes in the size of the disc when preinjection and three-month postinjection CT scans were compared. By six months, however, seven of ten patients (70%) had obvious changes in their CT scan. Seven patients (14%) were considered chymopapain treatment failures and were later treated with surgical discectomy. Only two of these seven patients (30%) had obvious changes in their three-month CT scan. Chymopapain injection did not alter the size of the herniated portion of the disc during the first three months after chymopapain injection. A decision to operate for presumed chymopapain failure should therefore be based on clinical grounds, rather than on the three-month CT appearance of the herniated disc.

Transection of the thoracic aorta: assessment of a trauma system.

A large, heavily populated area regionalized the care of critical trauma in 1980. To evaluate the system, we reviewed patient outcome for thoracic aortic transection due to blunt injury for the first 18 months of trauma system operation. Of the total of 86 patients, 43 were transferred to trauma centers, 8 to nontrauma centers, and 35 were either directly transported to the coroner or dead on arrival at the hospital. Of the eight patients transported to non-trauma centers, seven were in cardiopulmonary arrest during transport and the eighth was pronounced dead shortly after admission to the emergency department. Twenty-seven of the 43 patients transferred to trauma centers were dead within 24 minutes of admission. The cause of death was rupture of a transected aorta in 22 patients and other multiple injuries in the remaining 5. Sixteen were alive long enough in the emergency department for evaluation. Nine of these patients underwent correction of aortic transection as well as other injuries and all survived. Two of the nine survivors sustained partial or complete spinal cord damage. The remaining seven patients died, but in only one patient did the undiagnosed aortic injury contribute to the cause of death. This patient had a normal cineangiogram and the diagnosis was made at autopsy. He was considered potentially salvageable, so 9 of 10 potentially salvageable patients survived (90 percent). Of the total of 86 patients with aortic transection, 77 died (90 percent). This study shows that regionalization of trauma care offers an excellent chance for survival of patients with thoracic aortic transection.

Acquired methemoglobinemia and hemolytic anemia after usual doses of phenazopyridine.

Two patients developed symptomatic methemoglobinemia and hemolytic anemia after treatment with phenazopyridine. Methemoglobinemia appears to be a rare occurrence after commonly used doses of phenazopyridine; phenazopyridine-associated hemolytic anemia has been reported both after overdose and after usual doses. The presentation of methemoglobinemia in the first patient and the response to treatment with methylene blue in the second patient were unusual, suggesting that the patients had a red cell defect or were exposed to other oxidizing substances. One of the major metabolites of phenazopyridine is aniline, a known cause of methemoglobinemia. Aniline-induced methemoglobinemia is less responsive to treatment with methylene blue than nitrate- or nitrite-induced methemoglobinemia. This may explain, in part, the poor response to methylene blue by one of our patients.