Henry E. Rice

Neurogenic differentiation of murine and human adipose-derived stromal cells

The identification of cells capable of neuronal differentiation has great potential for cellular therapies. We examined whether murine and human adipose-derived adult stem (ADAS) cells can be induced to undergo neuronal differentiation. We isolated ADAS cells from the adipose tissue of adult BalbC mice or from human liposuction tissue and induced neuronal differentiation with valproic acid, butylated hydroxyanisole, insulin, and hydrocortisone. As early as 1-3 h after neuronal induction, the phenotype of ADAS cells changed towards neuronal morphology. Following neuronal induction, muADAS cells displayed immunocytochemical staining for GFAP, nestin and NeuN and huADAS cells displayed staining for intermediate filament M, nestin, and NeuN. Following neuronal induction of murine and human ADAS cells, Western blot analysis confirmed GFAP, nestin, and NeuN protein expression. Pretreatment with EGF and basic FGF augmented the neuronal differentiation of huADAS cells. The neuronal differentiation of stromal cells from adipose tissue has broad biological and clinical implications.

Characterization of neuronal/glial differentiation of murine adipose-derived adult stromal cells

Neural tissue has limited capacity for intrinsic repair after injury, and the identification of alternate sources of neuronal stem cells has broad clinical potential. Preliminary studies have demonstrated that adipose-derived adult stromal (ADAS) cells are capable of differentiating into mesenchymal and non-mesenchymal cells in vitro, including cells with select characteristics of neuronal/glial tissue. In this study, we extended these observations to test the hypothesis that murine (mu) ADAS cells can be induced to exhibit characteristics of neuronal and glial tissue by exposure to a cocktail of induction agents. We characterized the differentiation of muADAS cells in vitro using immunohistochemistry and immunoblotting, and examined whether these cells respond to the glutamate agonist N-methyl-D-aspartate (NMDA). We found that induced muADAS cells express proteins indicative of neuronal/glial cells, including nestin, GFAP, S-100, NeuN, MAP2, tau, and beta-III tubulin. Induced muADAS cells express gamma-aminobutyric acid (GABA), the NR-1 and NR-2 subunits of the glutamate receptor, GAP-43, synapsin I, and voltage-gated calcium channels. Finally, induced muADAS cells demonstrate decreased viability in response to NMDA. These findings suggest that muADAS cells can be induced to exhibit several phenotypic, morphologic, and excitotoxic characteristics consistent with developing neuronal and glial tissue.

Transplantation of Thymus Tissue in Complete DiGeorge Syndrome

BACKGROUND The DiGeorge syndrome is a congenital disorder that affects the heart, parathyroid glands, and thymus. In complete DiGeorge syndrome, patients have severely reduced T-cell function. METHODS We treated five infants (age, one to four months) with complete DiGeorge syndrome by transplantation of cultured postnatal thymus tissue. Follow-up evaluations included immune phenotyping and proliferative studies of peripheral-blood mononuclear cells plus biopsy of the thymus allograft. Thymic production of new T cells was assessed in peripheral blood by tests for T-cell-receptor recombination excision circles, which are formed from excised DNA during the rearrangement of T-cell-receptor genes. RESULTS After the transplantation of thymus tissue, T-cell proliferative responses to mitogens developed in four of the five patients. Two of the patients survived with restoration of immune function; three patients died from infection or abnormalities unrelated to transplantation. Biopsies of grafted thymus in the surviving patients showed normal morphologic features and active T-cell production. In three patients, donor T cells could be detected about four weeks after transplantation, although there was no evidence of graft-versus-host disease on biopsy or at autopsy. In one patient, the T-cell development within the graft was demonstrated to accompany the appearance of recently developed T cells in the periphery and coincided with the onset of normal T-cell function. In one patient, there was evidence of thymus function and CD45RA+CD62L+ T cells more than five years after transplantation. CONCLUSIONS In some infants with profound immunodeficiency and complete DiGeorge syndrome, the transplantation of thymus tissue can restore normal immune function. Early thymus transplantation - before the development of infectious complications - may promote successful immune reconstitution.

Long-term repopulating ability of xenogeneic transplanted human fetal liver hematopoietic stem cells in sheep.

We previously reported on the successful engraftment and long-term multilineage expression (erythroid, myeloid, lymphoid) of human fetal liver hematopoietic stem cells in sheep after transplantation in utero. That the engraftment of long-term repopulating pluripotent stem cells occurred in these animals was shown here by the fact that transplantation of human CD45+ cells isolated from bone marrow of these chimeric animals into preimmune fetal sheep resulted in engraftment and expression of human cells. Marrow cells were obtained from three chimeric sheep at 3.2-3.6 yr after transplant. The relative percentage of human CD45+ cells present in these marrows was 3.3 +/- 0.32%. A total of 29 x 10(6) CD45+ cells were isolated by panning, pooled, and transplanted into six preimmune sheep fetuses (4.8 x 10(6) cells/fetus). All six recipients were born alive. Hematopoietic progenitors exhibiting human karyotype were detected in marrows of two lambs soon after birth. Cells expressing human CD45 antigen were also detected in blood and marrow of both lambs. Human cell expression has been multilineage and has persisted for > 1 yr. These results demonstrate that the expression of human cells in this large animal model resulted from engraftment of long-term repopulating pluripotent human stem cells.

Congenital diaphragmatic hernia: a systematic review and summary of best-evidence practice strategies.

Objectives:Recent reports suggest that specific care strategies improve survival of infants with congenital diaphragmatic hernia (CDH). This review presents details of care from centers reporting high rates of survival among CDH infants.Study Design:We conducted a MEDLINE search (1995 to 2006) and searched all citations in the Cochrane Central Register of Controlled Trials. Studies were included if they contained reports of >20 infants with symptomatic CDH, and >75% survival of isolated CDH.Result:Thirteen reports from 11 centers met inclusion criteria. Overall survival, including infants with multiple anomalies, was 603/763 (79%; range: 69 to 93%). Survival for isolated CDH was 560/661 (85%; range: 78 to 96%). The frequency of extracorporeal membrane oxygenation (ECMO) use for isolated CDH varied widely among reporting centers 251/622 (40%; range: 11 to 61%), as did survival for infants with isolated CDH placed on ECMO: 149/206 (73%; range: 33 to 86%). There was no suggestion of benefit from use of antenatal glucocorticoids given after 34 weeks gestation or use of postnatal surfactant. Low mortality was frequently attributed to minimizing lung injury and adhering to center-specific criteria for ECMO.Conclusion:Use of strategies aimed at minimizing lung injury, tolerance of postductal acidosis and hypoxemia, and adhering to center-specific criteria for ECMO were strategies most consistently reported by successful centers. The literature lacks randomized clinical trials of these or other care strategies in this complex patient population; prospective studies of safety and long-term outcome are needed.

Clinical and Hematologic Benefits of Partial Splenectomy for Congenital Hemolytic Anemias in Children

ObjectiveTo assess the role of partial splenectomy for symptomatic children with various congenital hemolytic anemias. Summary Background DataThe use of total splenectomy for symptomatic children with congenital hemolytic anemias is restricted by concern of postsplenectomy sepsis. A partial splenectomy is an alternative procedure, although its utility remains incompletely defined. MethodsThis longitudinal cohort study followed 25 symptomatic children with various congenital anemias who underwent partial splenectomy. Sixteen children had hereditary spherocytosis (HS), and nine children had other erythrocyte disorders. Outcome measures were clinical and laboratory hemolysis, splenic phagocytic and immune function, and splenic regrowth as measured by ultrasonography. Discrete parameters were compared using the Student t test. ResultsPartial splenectomy was successful in all 25 children, with minimal morbidity. Follow-up ranged from 7 months to 6 years (mean 2.3 ± 1.5 years). Following surgery, children with HS had increased hemoglobin values, decreased reticulocyte and bilirubin levels, and preserved splenic function. Most children without HS had decreased symptoms of hypersplenism and splenic sequestration. Over time, variable rates of splenic regrowth were noted, although regrowth did not necessarily correlate with recurrent hemolysis. ConclusionsIn children with hereditary spherocytosis, a partial splenectomy appears to control hemolysis while retaining splenic function. In children with other congenital hemolytic anemias, a partial splenectomy appears to control symptoms of hypersplenism and splenic sequestration.

Evaluation of Nissen Fundoplication in Neurologically Impaired Children

The value of performing Nissen fundoplication in neurologically impaired children is a controversial issue. To evaluate the benefit of fundoplication in these children, hospital records were reviewed for 77 children who underwent fundoplication for gastroesophageal reflux (GER). Fifty-two children were neurologically impaired; 25 children had no neurological impairment. Impaired children had significantly fewer hospital admissions (1.8 v 0.7; P less than .005) and total days of hospitalization (36 v 14; P less than .005) during the first postoperative 6 months, compared with the immediate preoperative 6-month period. Normal children had fewer hospital admissions and days postoperatively, but the difference was not significant. Impaired children with preoperative failure to thrive (FTT had significantly increased average monthly weight gain over the first 6-month period postoperatively, compared with preoperative growth rate (3.0% v 0.9% of total body weight; P less than .05). Average monthly weight gain at 1 and 2 years postoperatively was not significantly different from preoperative values for impaired children. Growth rate of normal children with FTT did not change significantly postoperatively. Symptomatic relief was comparable in the normal and impaired children. Perioperative mortality was 0% in the normal children and 6% in the impaired children. This study demonstrates that Nissen fundoplication in neurologically impaired children with GER can be performed safely, reduces the frequency of hospitalization, and improves short-term weight gain.

Stem cell therapy for neurologic disorders: therapeutic potential of adipose-derived stem cells.

There is growing evidence to suggest that reservoirs of stem cells may reside in several types of adult tissue. These cells may retain the potential to transdifferentiate from one phenotype to another, presenting exciting possibilities for cellular therapies. Recent discoveries in the area of neural differentiation are particularly exciting given the limited capacity of neural tissue for intrinsic repair and regeneration. Adult adipose tissue is a rich source of mesenchymal stem cells, providing an abundant and accessible source of adult stem cells. These cells have been termed adipose derived stem cells (ASC). The characterization of these ASCs has defined a population similar to marrow-derived and skeletal muscle-derived stem cells. The success seen in differentiating ASC into various mesenchymal lineages has generated interest in using ASC for neuronal differentiation. Initial in vitro studies characterized the morphology and protein expression of ASC after exposure to neural induction agents. Additional in vitro data suggests the possibility that ASCs are capable of neuronal activity. Progress in the in vitro characterization of ASCs has led to in vivo modeling to determine the survival, migration, and engraftment of transplanted ASCs. While work to define the mechanisms behind the transdifferentiation of ASCs continues, their application to neurological diseases and injuries should also progress. The subject of this review is the capacity of adipose derived stem cells (ASC) for neural transdifferentiation and their application to the treatment of various neurologic disorders.

Does early ultrasonography affect management of pediatric appendicitis? A prospective analysis.

BACKGROUND Appendicitis remains a difficult diagnosis in children. Ultrasonography is increasingly used for the diagnosis of appendicitis, although the proper clinical role for this test remains unclear. METHODS To evaluate the clinical utility of ultrasonography in appendicitis, the authors analyzed prospectively all children evaluated for possible appendicitis from January 1 through December 31, 1997. Children with a high clinical suspicion of appendicitis were referred for surgery (n = 122). Children with equivocal findings of appendicitis were referred for early ultrasonography (EUS) and formed the study cohort (n = 103). An initial management plan was made to operate or observe each patient, and a risk of appendicitis (doubtful, possible, probable) was assigned by a pediatric surgery fellow. EUS was then performed, and its effect on management was assessed. RESULTS Using clinical judgment to operate at initial presentation, the sensitivity was 38% and specificity was 95%. Using EUS alone, the sensitivity was 87% and specificity was 88%. The management of 30 of 103 patients (30%) was changed after EUS, including a decision to operate in 28 patients and a decision not to operate in two patients. CONCLUSIONS EUS appears to have substantial clinical utility in children with equivocal findings of appendicitis, and its use complements the clinical management. The use of EUS can improve patient care and reduce hospital resource utilization.

Superparamagnetic Iron Oxide Labeling and Transplantation of Adipose-Derived Stem Cells in Middle Cerebral Artery Occlusion-Injured Mice

OBJECTIVE Adipose-derived stem cells are an alternative stem cell source for CNS therapies. The goals of the current study were to label adipose-derived stem cells with superparamagnetic iron oxide (SPIO) particles, to use MRI to guide the transplantation of adipose-derived stem cells in middle cerebral artery occlusion (MCAO)-injured mice, and to localize donor adipose-derived stem cells in the injured brain using MRI. We hypothesized that we would successfully label adipose-derived stem cells and image them with MRI. MATERIALS AND METHODS Adipose-derived stem cells harvested from mice inbred for green fluorescent protein were labeled with SPIO ferumoxide particles through the use of poly-L-lysine. Adipose-derived stem cell viability, iron staining, and proliferation were measured after SPIO labeling, and the sensitivity of MRI in the detection of SPIO-labeled adipose-derived stem cells was assessed ex vivo. Adult mice (n = 12) were subjected to unilateral MCAO. Two weeks later, in vivo 7-T MRI was performed to guide stereotactic transplantation of SPIO-labeled adipose-derived stem cells into brain tissue adjacent to the infarct. After 24 hours, the mice were sacrificed for high-resolution ex vivo 7-T or 9.4-T MRI and histologic study. RESULTS Adipose-derived stem cells were efficiently labeled with SPIO particles without loss of cell viability or proliferation. Using MRI, we guided precise transplantation of adipose-derived stem cells. MR images of mice given injections of SPIO-labeled adipose-derived stem cells had hypointense regions that correlated with the histologic findings in donor cells. CONCLUSION MRI proved useful in transplantation of adipose-derived stem cells in vivo. This imaging technique may be useful for studies of CNS stem cell therapies.