Azra Ahmed

Sternal repair with bone grafts engineered from amniotic mesenchymal stem cells

PURPOSE We aimed at determining whether osseous grafts engineered from amniotic mesenchymal stem cells (aMSCs) could be used in postnatal sternal repair. METHODS Leporine aMSCs were isolated, identified, transfected with green fluorescent protein (GFP), expanded, and seeded onto biodegradable electrospun nanofibrous scaffolds (n = 6). Constructs were dynamically maintained in an osteogenic medium and equally divided into 2 groups with respect to time in vitro as follows: 14.6 or 33.9 weeks. They were then used to repair full-thickness sternal defects spanning 2 to 3 intercostal spaces in allogeneic kits (n = 6). Grafts were submitted to multiple analyses 2 months thereafter. RESULTS Chest roentgenograms showed defect closure in all animals, confirmed at necropsy. Graft density as assessed by microcomputed tomographic scans increased significantly in vivo, yet there were no differences in mineralization by extracellular calcium measurements preimplantation and postimplantation. There was a borderline increase in alkaline phosphatase activity in vivo, suggesting ongoing graft remodeling. Histologically, implants contained GFP-positive cells and few mononuclear infiltrates. There were no differences between the 2 construct groups in any comparison. CONCLUSIONS Engineered osseous grafts derived from amniotic mesenchymal stem cells may become a viable alternative for sternal repair. The amniotic fluid can be a practical cell source for engineered chest wall reconstruction.

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.

Prenatal tracheal reconstruction with a hybrid amniotic mesenchymal stem cells–engineered construct derived from decellularized airway

PURPOSE This study was aimed at examining an airway construct engineered from autologous amniotic mesenchymal stem cells (aMSCs) and a xenologous decellularized airway scaffold as a means for tracheal repair. METHODS Fetal lambs (N = 13) with a tracheal defect were divided into 2 groups. One group (acellular, n = 6) was repaired with a decellularized leporine tracheal segment. The other group (engineered, n = 7) received an identical graft seeded with expanded/labeled autologous aMSCs. Newborns were euthanized for multiple analyses. RESULTS Eleven lambs survived to term, 10 of which could breathe at birth. Engineered grafts showed a significant increase in diameter in vivo (P = .04) unlike acellular grafts (P = .62), although variable stenosis was present in all implants. Engineered constructs exhibited full epithelialization, compared with none of the acellular grafts (P = .002). Engineered grafts had a significantly greater degree of increase in elastin levels after implantation than acellular implants (P = .04). No such differences were noted in collagen and glycosaminoglycan contents. Donor cells were detected in engineered grafts, which displayed a pseudostratified columnar epithelium. CONCLUSIONS Constructs engineered from aMSCs and decellularized airway undergo enhanced remodeling and epithelialization in vivo when compared with equivalent acellular implants. Amniotic mesenchymal stem cell-engineered airways may become an alternative for perinatal airway repair.

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.

Partial or complete coverage of experimental spina bifida by simple intra-amniotic injection of concentrated amniotic mesenchymal stem cells

PURPOSE We sought to determine whether simple intra-amniotic delivery of concentrated amniotic mesenchymal stem cells (afMSCs) may elicit prenatal coverage of experimental spina bifida. METHODS Time-dated pregnant Sprague-Dawley dams (n=24) exposed to retinoic acid for the induction of fetal neural tube defects were divided in three groups. Group I had no further manipulations. Groups II and III received volume-matched intra-amniotic injections of either saline (Group II) or a suspension of syngeneic afMSCs labeled with green fluorescent protein (Group III) in all fetuses (n=202) on gestational day 17 (term=21-22 days). Animals were killed before term. Statistical comparisons were by ANOVA (P<0.05). RESULTS Of 165 fetuses viable at euthanasia, a spina bifida was present in 58% (96/165), with no significant differences in defect dimension across the groups (P=0.19). However, variable degrees of coverage of the defect by a rudimentary skin confirmed histologically were only present in Group III (P<0.001), in which donor afMSCs were documented, with no differences between Groups I and II (P=0.98). CONCLUSIONS Amniotic mesenchymal stem cells can induce partial or complete coverage of experimental spina bifida after concentrated intra-amniotic injection. Trans-amniotic stem cell therapy (TRASCET) may become a practical option in the prenatal management of spina bifida.

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.

Trans-amniotic stem cell therapy (TRASCET) minimizes Chiari-II malformation in experimental spina bifida

PURPOSE We sought to study the impact of trans-amniotic stem cell therapy (TRASCET) in the Chiari-II malformation in experimental spina bifida. METHODS Sprague-Dawley fetuses (n=62) exposed to retinoic acid were divided into three groups at term (21-22 days gestation): untreated isolated spina bifida (n=21), isolated spina bifida treated with intra-amniotic injection of concentrated, syngeneic, labeled amniotic fluid mesenchymal stem cells (afMSCs) on gestational day 17 (n=28), and normal controls (n=13). Analyses included measurements of brainstem and cerebellar placement on high resolution MRI and histology. Statistical comparisons included ANOVA. RESULTS In parallel to the expected induced coverage of the spina bifida in the afMSC-treated group (P<0.001), there were statistically significant differences in brainstem displacement across the groups (P<0.001), with the highest caudal displacement in the untreated group. Significant differences in cerebellar displacement were also noted, albeit less pronounced. Pairwise comparisons were statistically significant, with P=0.014 between treated and normal controls in caudal brainstem displacement and P<0.001 for all other comparisons. Labeled afMSCs were identified in 71% of treated fetuses. CONCLUSIONS Induced coverage of spina bifida by TRASCET minimizes the Chiari-II malformation in the retinoic acid rodent model, further suggesting it as a practical alternative for the prenatal management of spina bifida.

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.

Targeted quantitative amniotic cell profiling: A potential diagnostic tool in the prenatal management of neural tube defects

PURPOSE We sought to determine whether amniotic cell profiles correlate quantitatively with neural tube defect (NTD) type and/or size. METHODS Sprague-Dawley fetuses exposed to retinoic acid (n=61) underwent amniotic fluid sample procurement before term. Samples were analyzed by flow cytometry for the presence of cells concomitantly expressing Nestin and Sox-2 (neural stem cells, aNSCs), and cells concomitantly expressing CD29 and CD44 (mesenchymal stem cells, aMSCs). Statistical analysis included ANOVA and post-hoc Bonferroni adjusted comparisons (P<0.05). RESULTS There was a statistically significant increase in the proportion of aNSCs in fetuses with spina bifida (6.78%± 1.87%) when compared to those with exencephaly (0.64%± 0.23%) or with both spina bifida and exencephaly (0.22%± 0.09%). Conversely, there was a statistically significant decrease in the proportion of aMSCs in fetuses with exencephaly, either isolated (1.09%± 0.42%) or in combination defects (2.37%± 0.63%) when compared with normal fetuses (8.83%± 1.38%). In fetuses with isolated exencephaly, there was a statistically significant inverse correlation between the proportion of aNSCs and defect size. CONCLUSIONS The proportions of neural and mesenchymal stem cells in the amniotic fluid correlate with the type and size of experimental NTDs. Targeted quantitative amniotic cell profiling may become a useful diagnostic tool in the prenatal evaluation of these anomalies.

Intra-Amniotic Delivery of Amniotic-Derived Neural Stem Cells in a Syngeneic Model of Spina Bifida

Objective: Neural stem cells (NSCs) may promote spinal cord repair in fetuses with experimental spina bifida. We sought to determine the fate of amniotic-derived NSCs (aNSCs) after simple intra-amniotic injection in a syngeneic model of spina bifida. Methods: Fetal neural tube defects were induced on 20 pregnant Lewis dams by prenatal administration of retinoic acid. Ten dams served as amniotic fluid donors for epigenetic isolation of aNSCs, which were expanded and labeled with 5-bromo-2′-deoxyuridine. The remaining 10 dams received intra-amniotic injections of the processed aNSCs, blindly in all their fetuses (n = 37) on gestational day 17 (term = E21-22). Fetuses with spina bifida underwent screening for the presence of donor aNSCs in the spinal cord at term. Results: Donor cells were identified in 93.3% of the animals with spina bifida, selectively populating the neural placode, typically in clusters, retaining an undifferentiated morphology, and predominantly on exposed neural surfaces, though some were detected deeper in neighboring neural tissue. Conclusions: The amniotic cavity can serve as a route of administration of NSCs in experimental spina bifida. Simple intra-amniotic delivery of NSCs may be a practical adjuvant to regenerative strategies for the treatment of spina bifida.