Justin D. Klein

Preclinical regulatory validation of an engineered diaphragmatic tendon made with amniotic mesenchymal stem cells

PURPOSE Under a Food and Drug Administration directive, we examined definite long-term safety and efficacy aspects of an engineered diaphragmatic tendon graft as a regulatory prerequisite for clinical trials. METHODS Newborn lambs (N = 27) underwent partial diaphragmatic replacement with a Teflon patch, a composite acellular bioprosthesis, or the same bioprosthesis seeded with autologous amniotic mesenchymal stem cells processed under Good Manufacturing Practice guidelines. Multiple safety and efficacy analyses were performed at different time points up to 14 months of age (ovine adulthood). RESULTS There was no mortality. None of the blood tests or full body autopsy specimens showed any abnormality. There was a significantly higher failure rate in animals that received an acellular bioprosthetic graft vs an engineered graft, with no significant differences between Teflon and acellular bioprosthetic implants. Tensile strength and total collagen levels were significantly higher in engineered grafts than in acellular bioprosthetic grafts. On histology, lysozyme and myeloperoxidase stainings were unremarkable in all grafts. CONCLUSIONS Diaphragmatic repair with a clinically viable autologous tendon engineered with amniotic mesenchymal stem cells leads to improved outcomes when compared with an equivalent acellular bioprosthesis, with no local or systemic adverse effects. Clinical trials of engineered diaphragmatic repair appear practicable within regulatory guidelines.

Amniotic Mesenchymal Stem Cells Enhance Normal Fetal Wound Healing

Fetal wound healing involves minimal inflammation and limited scarring. Its mechanisms, which remain to be fully elucidated, hold valuable clues for wound healing modulation and the development of regenerative strategies. We sought to determine whether fetal wound healing includes a hitherto unrecognized cellular component. Two sets of fetal lambs underwent consecutive experiments at midgestation. First, fetuses received an intra-amniotic infusion of labeled autologous amniotic mesenchymal stem cells (aMSCs), in parallel to different surgical manipulations. Subsequently, fetuses underwent creation of 2 symmetrical, size-matched skin wounds, both encased by a titanium chamber. One of the chambers was left open and the other covered with a semipermeable membrane that allowed for passage of water and all molecules, but not any cells. Survivors from both experiments had their wounds analyzed at different time points before term. Labeled aMSCs were documented in all concurrent surgical wounds. Covered wounds showed a significantly slower healing rate than open wounds. Paired comparisons indicated significantly lower elastin levels in covered wounds at the mid time points, with no significant differences in collagen levels. No significant changes in hyaluronic acid levels were detected between the wound types. Immunohistochemistry for substance P was positive in both open and covered wounds. We conclude that fetal wound healing encompasses an autologous yet exogenous cellular component in naturally occurring aMSCs. Although seemingly not absolutely essential to the healing process, amniotic cells expedite wound closure and enhance its extracellular matrix profile. Further scrutiny into translational implications of this finding is warranted.

Amniotic and Placental Mesenchymal Stem Cell Isolation and Culture

The amniotic fluid and placenta are sources of diverse progenitor cell populations, including -mesenchymal, hematopoietic, trophoblastic, and possibly more primitive stem cells. Given that much of the amniotic cavity and placenta share a common origin, namely the inner cell mass of the morula, perhaps it is not surprising that most types of progenitor cells that can be isolated from these two sources also share many characteristics. This chapter focuses solely on the most abundant and easy to isolate progenitor cell population found therein, the mesenchymal stem cells (MSCs). Unlike some of the other stem cell types, MSCs are present throughout gestation. Methods of isolation, expansion, freezing, and thawing of these cells will be presented with preference given to the simplest methods available for any given procedure.

Chest wall repair with engineered fetal bone grafts: an efficacy analysis in an autologous leporine model.

PURPOSE We sought to compare the efficacy of engineered fetal bone grafts with acellular constructs in an autologous model of chest wall repair. METHODS Rabbits (n = 10) with a full-thickness sternal defect were equally divided in 2 groups based on how the defect was repaired, namely, either with an autologous bone construct engineered with amniotic mesenchymal stem cells on a nanofibrous scaffold or a size-matched identical scaffold with no cells. Animals were killed at comparable time-points 18 to 20 weeks postimplantation for multiple analyses. RESULTS Gross evidence of nonunion confirmed by micro-computed tomography scanning was present in 3 (60%) of 5 of the acellular implants but in no engineered grafts. Histology confirmed the presence of bone in both types of repair, albeit seemingly less robust in the acellular grafts. Mineral density in vivo was significantly higher in engineered grafts than in acellular ones, with more variability among the latter. There was no difference in alkaline phosphatase activity between the groups. CONCLUSIONS Chest wall repair with an autologous osseous graft engineered with amniotic mesenchymal stem cells leads to improved and more consistent outcomes in the midterm when compared with an equivalent acellular prosthetic repair in a leporine model. Amniotic fluid-derived engineered bone may become a practical alternative for perinatal chest wall reconstruction.

The Amniotic Fluid As a Source of Neural Stem Cells in the Setting of Experimental Neural Tube Defects

We sought to determine whether neural stem cells (NSCs) can be isolated from the amniotic fluid in the setting of neural tube defects (NTDs), as a prerequisite for eventual autologous perinatal therapies. Pregnant Sprague-Dawley dams (n=62) were divided into experimental (n=42) and control (n=20) groups, depending on prenatal exposure to retinoic acid for the induction of fetal NTDs. Animals were killed before term for analysis (n=685 fetuses). Amniotic fluid samples from both groups underwent epigenetic selection for NSCs, followed by exposure to neural differentiation media. Representative cell samples underwent multiple morphological and phenotypical analyses at different time points. No control fetus (n=267) had any structural abnormality, whereas at least one type of NTD developed in 52% (217/418) of the experimental fetuses (namely, isolated spina bifida, n=144; isolated exencephaly, n=24; or a combination of the two, n=49). Only amniotic samples from fetuses with a NTD yielded cells with typical neural progenitor morphology and robust expression of both Nestin and Sox-2, primary markers of NSCs. These cells responded to differentiation media by displaying typical morphological changes, along with expression of beta-tubulin III, glial fibrillary acidic protein, and/or O4, markers for immature neurons, astrocytes, and oligodendrocytes, respectively. This was concurrent with downregulation of Nestin and Sox-2. We conclude that the amniotic fluid can harbor disease-specific stem cells, for example, NSCs in the setting of experimental NTDs. The amniotic fluid may be a practical source of autologous NSCs applicable to novel forms of therapies for spina bifida.

Adrenal cortical tumors in children: factors associated with poor outcome

PURPOSE The purpose of this study was to evaluate recurrence and survival outcomes in pediatric adrenal cortical neoplasms. METHODS A 90-year retrospective review of children with adrenal cortical neoplasms was performed using multivariate Cox regression analysis to identify factors associated with recurrence and tumor-related mortality. RESULTS The evaluable cohort included 29 patients. Twenty-seven underwent resection. Twenty-two (81%) had localized disease, and 5 (19%) had locally advanced disease (all received chemotherapy and 2 of 5 were cured). Two patients presenting with metastatic disease died despite treatment. There were 4 recurrences; all patients died. Tumor-related mortality was 24% (7/29). Kaplan-Meier freedom from recurrence was 85% at 1 year (95% confidence interval, 75%-95%). Multivariate Cox regression revealed that older age (P = .01), higher mitotic rate (P = .005), and necrosis (P < .001) were independent predictors of tumor-related death. Higher mitotic rate (P = .007) and larger tumor size (P = .03) were significant predictors of tumor recurrence. CONCLUSION Risk factors for poor outcomes in patients with adrenocortical tumors include older age, higher mitotic rate, higher percent necrosis, and larger tumor size. Therefore, the presence of these factors may warrant consideration of adjuvant chemotherapy, even in the absence of advanced disease.

Craniofacial repair with fetal bone grafts engineered from amniotic mesenchymal stem cells

BACKGROUND Ethically acceptable applications of fetal tissue engineering as a perinatal therapy can be expanded beyond life-threatening anomalies by amniotic fluid cell-based methods, in which cell procurement poses no additional risk to the mother. We sought to start to determine whether osseous grafts engineered from amniotic mesenchymal stem cells (aMSCs) could be an adjunct to craniofacial repair. METHODS New Zealand rabbits (n = 12) underwent creation of a full-thickness diploic nasal bone defect. We then equally divided animals into two groups based on how the defect was repaired: namely, size-matched implants of electrospun biodegradable nanofibers with or without nuclear labeled, allogeneic aMSCs maintained in osteogenic medium. We killed animals 8 wk post-implantation for multiple analyses. Statistical analysis included analysis of variance, post-hoc Bonferroni adjusted comparisons, and Levenes F-test, as appropriate (P < 0.05), with significance set at P < 0.05. RESULTS Micro-computed tomography scanning (two- and three-dimensional) showed no significant differences in defect radiodensity between groups. However, extracellular calcium levels were significantly higher in engineered grafts than in acellular implants (P = 0.003). There was significantly greater variability in mineralization in acellular implants than in engineered grafts by both direct calcium (P = 0.008) and micro-computed tomography measurements (P = 0.032). There were no significant differences in alkaline phosphatase activity or variance between groups. We documented labeled cells in the engineered grafts. CONCLUSIONS Craniofacial repair with osseous grafts engineered from aMSCs lead to enhanced and more consistent mineralization compared with an equivalent acellular prosthetic repair. Amniotic fluid-derived engineered bone may become a practical adjunct to perinatal craniofacial reconstruction.

Operative indications in recurrent ileocolic intussusception

BACKGROUND Air-contrast enema (ACE) is standard treatment for primary ileocolic intussusception. Management of recurrences is less clear. This study aimed to delineate appropriate therapy by quantifying the relationship between recurrence and need for bowel resection, pathologic lead points (PLP), and complication rates. METHODS After IRB approval, a single institution review of patients with ileocolic intussusception from 1997 to 2013 was performed, noting recurrences, outcomes, and complications. Fishers exact and t-tests were used. RESULTS Of 716 intussusceptions, 666 were ileocecal. Forty-four underwent bowel resection, with 29 PLPs and 9 ischemia/perforation. Recurrence after ACE occurred in 96 (14%). Recurrence did not predict PLP (P=0.25). Recurrence (≥3) was associated with higher resection rate (P=0.03), but not ischemia/perforation (P=0.75). ACE-related complications occurred in 4 (0.5%) patients. Successful initial ACE had 98% negative predictive value for resection and PLP (e.g., after successful ACE, 2% had resections, 2% PLP). After failed initial ACE, 36% received resection, and 23% had PLP (P<0.001). CONCLUSIONS Recurrence is associated with a greater risk of resection but not PLP or ACE-complication. Failed ACE is associated with increased risk for harboring PLP and receiving resection. ACE should be the standard treatment in recurrent intussusception, regardless of number of recurrences.

A large animal model of the fetal tracheal stenosis/atresia spectrum.

BACKGROUND Treatment of congenital tracheal stenosis/atresia remains essentially unresolved. Previous models of this disease entity have been restricted to rodents and the chick. We sought to establish the principles of a large, surgical animal model of this spectrum of fetal anomalies. METHODS Fetal lambs (n = 8) underwent open surgery at 90-112 days gestation. Their cervical tracheas were encircled by a biocompatible polytetrafluoroethylene wrap, so as to extrinsically restrict their external diameter by 25%. Survivors (n = 7) were killed at different time points post-operatively before term. The manipulated tracheal segments were compared with their respective proximal portions (controls). Analyses included morphometry, histology and quantitative extracellular matrix measurements. RESULTS At necropsy, the typical gross appearance of tracheal stenosis/atresia was present in all manipulated tracheal segments. Histological findings included the virtual disappearance of the membranous portion of the trachea, along with infolding, fragmentation, and/or posterior fusion of cartilaginous rings, often with disappearance of the airway mucosa. There were significant decreases in diameter (P < 0.001) and total collagen levels (P = 0.005) on the manipulated trachea compared with the control portions. No significant differences were observed in overall elastin or glycosaminoglycan contents. A significant time-dependent increase in elastin was noted on the control, but not the experimental side. CONCLUSIONS In a surgical ovine model, controlled extrinsic compression of the fetal trachea leads to morphological and biochemical findings compatible with the congenital tracheal stenosis/atresia spectrum. This simple and easily reproducible prenatal model can be instrumental in the development of emerging therapies for these congenital anomalies.

Familial case of prenatally diagnosed intralobar and extralobar sequestrations with cystadenomatoid change

Hybrid lesions are part of a spectrum of rare pulmonary diseases that are characterized as having elements of both congenital pulmonary airway malformation and bronchopulmonary sequestration. Fetal thoracic masses arise from alterations during lung development that are separated by timing of the inciting event and are often associated with an underlying degree of bronchial atresia. There are a handful of documented reports of sequestrations occurring in siblings, but no known reports of prenatally diagnosed lesions occurring in families. We present a case of 2 siblings diagnosed prenatally with fetal thoracic lesions who underwent postnatal resection revealing hybrid lesions on pathologic examination. Newer radiologic techniques have increased our ability to detect these masses prenatally, as well as follow them throughout pregnancy to determine their natural history. Ongoing laboratory investigation into the etiology of congenital lung lesions has brought forth more questions and suggested a familial component at a cellular level that has not yet been fully discovered. We reviewed the current literature of factors contributing to the development of congenital lung lesions and suggest that there is a familial link in certain patient populations where screening may be indicated.