Mahesh Choolani

Superior osteogenic capacity for bone tissue engineering of fetal compared with perinatal and adult mesenchymal stem cells

Mesenchymal stem cells (MSCs) from human adult bone marrow (haMSCs) represent a promising source for bone tissue engineering. However, their low frequencies and limited proliferation restrict their clinical utility. Alternative postnatal, perinatal, and fetal sources of MSCs appear to have different osteogenic capacities, but have not been systematically compared with haMSCs. We investigated the proliferative and osteogenic potential of MSCs from human fetal bone marrow (hfMSCs), human umbilical cord (hUCMSCs), and human adult adipose tissue (hATMSCs), and haMSCs, both in monolayer cultures and after loading into three‐dimensional polycaprolactone‐tricalcium‐phosphate scaffolds.Although all MSCs had comparable immunophenotypes, only hfMSCs and hUCMSCs were positive for the embryonic pluripotency markers Oct‐4 and Nanog. hfMSCs expressed the lowest HLA‐I level (55% versus 95%–99%) and the highest Stro‐1 level (51% versus 10%–27%), and had the greatest colony‐forming unit–fibroblast capacity (1.6×–2.0×; p < .01) and fastest doubling time (32 versus 54–111 hours; p < .01). hfMSCs had the greatest osteogenic capacity, as assessed by von‐Kossa staining, alkaline phosphatase activity (5.1×–12.4×; p < .01), calcium deposition (1.6×–2.7× in monolayer and 1.6×–5.0× in scaffold culture; p < .01), calcium visualized on micro‐computed tomography (3.9×17.6×; p < .01) and scanning electron microscopy, and osteogenic gene induction. Two months after implantation of cellular scaffolds in immunodeficient mice, hfMSCs resulted in the most robust mineralization (1.8×–13.3×; p < .01).The ontological and anatomical origins of MSCs have profound influences on the proliferative and osteogenic capacity of MSCs. hfMSCs had the most proliferative and osteogenic capacity of the MSC sources, as well as being the least immunogenic, suggesting they are superior candidates for bone tissue engineering. STEM CELLS 2009;27:126–137

Functional genomics identifies five distinct molecular subtypes with clinical relevance and pathways for growth control in epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is hallmarked by a high degree of heterogeneity. To address this heterogeneity, a classification scheme was developed based on gene expression patterns of 1538 tumours. Five, biologically distinct subgroups — Epi‐A, Epi‐B, Mes, Stem‐A and Stem‐B — exhibited significantly distinct clinicopathological characteristics, deregulated pathways and patient prognoses, and were validated using independent datasets. To identify subtype‐specific molecular targets, ovarian cancer cell lines representing these molecular subtypes were screened against a genome‐wide shRNA library. Focusing on the poor‐prognosis Stem‐A subtype, we found that two genes involved in tubulin processing, TUBGCP4 and NAT10, were essential for cell growth, an observation supported by a pathway analysis that also predicted involvement of microtubule‐related processes. Furthermore, we observed that Stem‐A cell lines were indeed more sensitive to inhibitors of tubulin polymerization, vincristine and vinorelbine, than the other subtypes. This subtyping offers new insights into the development of novel diagnostic and personalized treatment for EOC patients.

Unsupervised High-Dimensional Analysis Aligns Dendritic Cells across Tissues and Species.

Summary Dendritic cells (DCs) are professional antigen-presenting cells that hold great therapeutic potential. Multiple DC subsets have been described, and it remains challenging to align them across tissues and species to analyze their function in the absence of macrophage contamination. Here, we provide and validate a universal toolbox for the automated identification of DCs through unsupervised analysis of conventional flow cytometry and mass cytometry data obtained from multiple mouse, macaque, and human tissues. The use of a minimal set of lineage-imprinted markers was sufficient to subdivide DCs into conventional type 1 (cDC1s), conventional type 2 (cDC2s), and plasmacytoid DCs (pDCs) across tissues and species. This way, a large number of additional markers can still be used to further characterize the heterogeneity of DCs across tissues and during inflammation. This framework represents the way forward to a universal, high-throughput, and standardized analysis of DC populations from mutant mice and human patients.

An EMT spectrum defines an anoikis-resistant and spheroidogenic intermediate mesenchymal state that is sensitive to e-cadherin restoration by a src-kinase inhibitor, saracatinib (AZD0530)

The phenotypic transformation of well-differentiated epithelial carcinoma into a mesenchymal-like state provides cancer cells with the ability to disseminate locally and to metastasise. Different degrees of epithelial–mesenchymal transition (EMT) have been found to occur in carcinomas from breast, colon and ovarian carcinoma (OC), among others. Numerous studies have focused on bona fide epithelial and mesenchymal states but rarely on intermediate states. In this study, we describe a model system for appraising the spectrum of EMT using 43 well-characterised OC cell lines. Phenotypic EMT characterisation reveals four subgroups: Epithelial, Intermediate E, Intermediate M and Mesenchymal, which represent different epithelial–mesenchymal compositions along the EMT spectrum. In cell-based EMT-related functional studies, OC cells harbouring an Intermediate M phenotype are characterised by high N-cadherin and ZEB1 expression and low E-cadherin and ERBB3/HER3 expression and are more anoikis-resistant and spheroidogenic. A specific Src-kinase inhibitor, Saracatinib (AZD0530), restores E-cadherin expression in Intermediate M cells in in vitro and in vivo models and abrogates spheroidogenesis. We show how a 33-gene EMT Signature can sub-classify an OC cohort into four EMT States correlating with progression-free survival (PFS). We conclude that the characterisation of intermediate EMT states provides a new approach to better define EMT. The concept of the EMT Spectrum allows the utilisation of EMT genes as predictive markers and the design and application of therapeutic targets for reversing EMT in a selective subgroup of patients.

Neo-vascularization and bone formation mediated by fetal mesenchymal stem cell tissue-engineered bone grafts in critical-size femoral defects

Tissue-engineered bone grafts (TEBG) require highly osteogenic cell sources for use in fracture repair applications. Compared to other sources of mesenchymal stem cells (MSC), human fetal MSC (hfMSC) have recently been shown to be more proliferative and osteogenic. We studied the functional performance of hfMSC-mediated TEBG in 7 mm rat femoral critical-sized bone defects (CSD). Dynamically-cultured and osteogenically-primed hfMSC seeded onto macroporous poly-epsilon-caprolactone tri-calcium phosphate scaffolds were transplanted into CSDs. After 12 weeks, hfMSC-mediated TEBG induced 2.1x more new bone formation (43.3+/-10.5 vs. 21.0+/-7.4 mm(3), p<0.05), with greater compact and woven bone, and a 9.8x increase in stiffness (3.9+/-1.7 vs. 0.4+/-0.3 mNm/degree, p<0.05) compared to acellular scaffolds, such that only animals transplanted with TEBG underwent full fracture repair of the CSD. Although hfMSC survived for <4 weeks, by 4 weeks they were associated with a 3.9x larger vasculature network in the defect area (35.2+/-11.1 vs. 6.5+/-3.6 mm(3)p<0.05), suggesting an important role for hfMSC in the promotion of neo-vasculogenesis. We speculate that hfMSC-mediated healing of the CSD by stimulating neo-vascularization through as yet undetermined mechanisms. This proof-of-principle study demonstrates the utility of primitive MSC for bone regeneration, and may be of relevance to vascularization in other areas of regenerative medicine.

A biaxial rotating bioreactor for the culture of fetal mesenchymal stem cells for bone tissue engineering.

The generation of effective tissue engineered bone grafts requires efficient exchange of nutrients and mechanical stimulus. Bioreactors provide a manner in which this can be achieved. We have recently developed a biaxial rotating bioreactor with efficient fluidics through in-silico modeling. Here we investigated its performance for generation of highly osteogenic bone graft using polycaprolactone-tricalcium phosphate (PCL-TCP) scaffolds seeded with human fetal mesenchymal stem cell (hfMSC). hfMSC scaffolds were cultured in either bioreactor or static cultures, with assessment of cellular viability, proliferation and osteogenic differentiation in vitro and also after transplantation into immunodeficient mice. Compared to static culture, bioreactor-cultured hfMSC scaffolds reached cellular confluence earlier (day 7 vs. day 28), with greater cellularity (2x, p<0.01), and maintained high cellular viability in the core, which was 2000 microm from the surface. In addition, bioreactor culture was associated with greater osteogenic induction, ALP expression (1.5x p<0.01), calcium deposition (5.5x, p<0.001) and bony nodule formation on SEM, and in-vivo ectopic bone formation in immunodeficient mice (3.2x, p<0.001) compared with static-cultured scaffolds. The use of biaxial bioreactor here allowed the maintenance of cellular viability beyond the limits of conventional diffusion, with increased proliferation and osteogenic differentiation both in vitro and in vivo, suggesting its utility for bone tissue engineering applications.

Microcarrier culture for efficient expansion and osteogenic differentiation of human fetal mesenchymal stem cells.

Abstract Stirred microcarrier (MC) culture has been suggested as the method of choice for supplying large volumes of mesenchymal stem cells (MSCs) for bone tissue engineering. In this study, we show that in addition to the improvement in cell expansion capacity, MSCs propagated and harvested from MC culture also demonstrate higher osteogenic potency when differentiated in vivo or in vitro in three-dimensional (3D) scaffold cultures as compared with traditional monolayer (MNL) cultures. Cytodex 3 microcarrier-expanded human fetal MSC (hfMSC) cultures (MC-hfMSCs) achieved 12- to 16-fold expansion efficiency (6×105–8×105 cells/mL) compared to 4- to 6-fold (1.2×105–1.8×105 cells/mL) achieved by traditional MNL-expanded hfMSC culture (MNL-hfMSCs; p<0.05). Both MC-hfMSCs and MNL-hfMSCs maintained similar colony-forming capacity, doubling times, and immunophenotype postexpansion. However, when differentiated under in vitro two-dimensional (2D) osteogenic conditions, MC-hfMSCs exhibited a 45-fold reduction in alkaline phosphatase level and a 37.5% decrease in calcium deposition compared with MNL-hfMSCs (p<0.05). Surprisingly, when MC-hfMSCs and MNL-hfMSCs were seeded on 3D macroporous scaffold culture or subcutaneously implanted into nonobese diabetic/severe combined immunodeficient mice, MC-hfMSCs deposited 63.5% (p<0.05) more calcium and formed 47.2% (p<0.05) more bone volume, respectively. These results suggest that the mode of hfMSC growth in the expansion phase affects the osteogenic potential of hfMSCs differently in various differentiation platforms. In conclusion, MC cultures are advantageous over MNL cultures in bone tissue engineering because MC-hfMSCs have improved cell expansion capacity and exhibit higher osteogenic potential than MNL-hfMSCs when seeded in vitro into 3D scaffolds or implanted in vivo.

A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering

Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4-2.5x, p < 0.05) in large 785 mm(3) macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2-1.6×, p < 0.01) and calcium deposition (1.3-2.3×, p < 0.01). We developed a Micro CT quantification method which demonstrated homogenous distribution of hfMSC in BXR bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds.

Quantitative Proteomics Analysis of Maternal Plasma in Down Syndrome Pregnancies Using Isobaric Tagging Reagent (iTRAQ)

Currently no specific biomarkers exist for the screening of pregnancies at risk for down syndrome (DS). Since a quantitative proteomic approach with isobaric labelling (iTRAQ) has recently been suggested to be highly suitable for the discovery of novel plasma biomarkers, we have now used this method to examine for potential quantitative changes in the plasma proteome of the pregnancies bearing DS fetuses in comparison to normal healthy babies. In our study, we used plasma from six women with DS pregnancies and six with uncomplicated pregnancies care were taken to match cases and controls for gestational and maternal age, as these could be a confounder. In our quantitative proteomics analysis we were able to detect 178 proteins using iTRAQ labelling in conjunction with 4800 MALDI TOF/TOF. Amongst these we observed changes in βHCG, a known screening marker for DS, indicating that our assay was functional. We found a number of elevated proteins Ig lambda chain C region, serum amyloid P-component, amyloid beta A4, and under expressed proteins like gamma-actin and titin in DS pregnancies. These proteins are also found in the sera of patients with Alzheimer disease, which share similar pathologies of DS. Our study therefore indicates that the iTRAQ labelling approach may be indeed useful for the detection of novel biomarkers.

Pre- and Postnatal Transplantation of Fetal Mesenchymal Stem Cells in Osteogenesis Imperfecta: A Two-Center Experience

Osteogenesis imperfecta (OI) can be recognized prenatally with ultrasound. Transplantation of mesenchymal stem cells (MSCs) has the potential to ameliorate skeletal damage. We report the clinical course of two patients with OI who received prenatal human fetal MSC (hfMSC) transplantation and postnatal boosting with same‐donor MSCs. We have previously reported on prenatal transplantation for OI type III. This patient was retransplanted with 2.8 × 106 same‐donor MSCs per kilogram at 8 years of age, resulting in low‐level engraftment in bone and improved linear growth, mobility, and fracture incidence. An infant with an identical mutation who did not receive MSC therapy succumbed at 5 months despite postnatal bisphosphonate therapy. A second fetus with OI type IV was also transplanted with 30 × 106 hfMSCs per kilogram at 31 weeks of gestation and did not suffer any new fractures for the remainder of the pregnancy or during infancy. The patient followed her normal growth velocity until 13 months of age, at which time longitudinal length plateaued. A postnatal infusion of 10 × 106 MSCs per kilogram from the same donor was performed at 19 months of age, resulting in resumption of her growth trajectory. Neither patient demonstrated alloreactivity toward the donor hfMSCs or manifested any evidence of toxicities after transplantation. Our findings suggest that prenatal transplantation of allogeneic hfMSCs in OI appears safe and is of likely clinical benefit and that retransplantation with same‐donor cells is feasible. However, the limited experience to date means that it is not possible to be conclusive and that further studies are required.