Lonnie D. Shea

The cryopreservation and autotransplantation of ovarian tissue is emerging as a powerful approach for preserving fertility. However, for cancer patients, it may not be possible to transplant ovarian tissue due to the risk of re-seeding disease. We investigated strategies for transplantation of individually isolated follicles to minimize the risk of re-introducing cancer cells present within the vasculature of ovarian stroma. Procedures for large-scale isolation of early-stage follicles and their encapsulation into fibrin hydrogels were developed. For in vivo validation studies, mice were ovariectomized and transplanted with encapsulated follicles into the ovarian bursa. A substantial increase in the number of secondary follicles was observed in the graft at 9 days after transplantation, and antral follicles by day 21, demonstrating primordial follicle recruitment into the growing pool. Initially, elevated follicle-stimulating hormone levels declined substantially by day 21, indicating feedback from the graft; presence of corpora lutea showed the graft's capability of restoring hormone cyclicity. Taken together, the transplanted follicles were able to engraft, mature, and restore ovarian function in an infertile mouse. This biomaterial may, thus, provide a platform for follicle transplantation with a low risk of cancer contamination and for developing strategies that preserve fertility for women facing a cancer diagnosis.

Chemo- and radiation therapies used to treat cancer can have the unintended effect of making patients infertile. Clinically established fertility preservation methods, such as egg and embryo cryopreservation, are not applicable to all patients, which has motivated the development of strategies that involve ovarian tissue removal and cryopreservation before the first sterilizing treatment. To restore fertility at a later date, the early-stage follicles present in the tissue must be matured to produce functional oocytes, a process that is not possible using existing cell culture technologies. This review describes the application of tissue engineering principles to promote ovarian follicle maturation and produce mature oocytes through either in vitro culture or transplantation. The design principles for these engineered systems are presented, along with identification of emerging opportunities in reproductive biology.

Particle-based technologies are increasingly being used in diagnostics and therapeutics. The particles employed in these applications are usually composed of polymers such as poly(lactide-co-glycolide) (PLG) and functionalized with peptides or proteins. Peptide or protein conjugation to particles is frequently achieved using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), while dimethyl sulfoxide (DMSO) is used to retrieve surface-attached or encapsulated peptides or proteins by solubilizing the particles. We examined strategies based on bicinchoninic acid (BSA), Coomassie Plus, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays for the quantification of surface-attached or encapsulated peptides or proteins. We determined that the CBQCA assay is a highly sensitive and accurate substitute for radioactivity-based assays that is suitable for measuring multiple particle-bound or particle-encapsulated peptides or proteins in the presence of EDC or PLG in DMSO, compounds that interfere with ...

A biomaterial-mediated gene therapy was used to activate local populations of oligodendrocytes progenitors and enhance regeneration after spinal cord injury.

Hydrogels, which have become a central component of numerous strategies in regenerative medicine, have recently been designed to include pores as a means to facilitate cell ingrowth and facilitate transport. Herein, we present the formation of macro-porous hydrogels by a novel fabrication method termed cryotemplated photopolymerization. In contrast to chemically-induced cryogelation, our cryotemplation method separates the creation of pores from the crosslinking of the polymer, which allows templating of hydrogels using both porogens and light. This method allows separately frozen pieces to be joined during the photopolymerization, without the use of a mold, to form complex architectures. The size of the pores in the hydrogels could be controlled by multiple methods, thus providing a versatile platform for numerous tissue engineering applications. Additionally, these hydrogels were capable of functionalization with peptides using techniques that did not interfere with gelation. Furthermore, porous hydrogels could be formed under conditions suitable for cell freezing thereby allowing for cell encapsulation. These studies characterize a hydrogel fabrication strategy that enables the creation of porous scaffolds in complex architectures, while retaining the potential to chemically functionalize the hydrogels.

The directed differentiation toward erythroid (E) or megakaryocytic (MK) lineages by the MK‐E progenitor (MEP) could enhance the ex vivo generation of red blood cells and platelets for therapeutic transfusions. The lineage choice at the MEP bifurcation is controlled in large part by activity within the intracellular signal transduction network, the output of which determines the activity of transcription factors (TFs) and ultimately gene expression. Although many TFs have been implicated, E or MK differentiation is a complex process requiring multiple days, and the dynamics of TF activities during commitment and terminal maturation are relatively unexplored. Herein, we applied a living cell array for the large‐scale, dynamic quantification of TF activities during MEP bifurcation. A panel of hematopoietic TFs (GATA‐1, GATA‐2, SCL/TAL1, FLI‐1, NF‐E2, PU.1, c‐Myb) was characterized during E and MK differentiation of bipotent K562 cells. Dynamic TF activity profiles associated with differentiation towards each lineage were identified, and validated with previous reports. From these activity profiles, we show that GATA‐1 is an important hub during early hemin‐ and PMA‐induced differentiation, and reveal several characteristic TF interactions for E and MK differentiation that confirm regulatory mechanisms documented in the literature. Additionally, we highlight several novel TF interactions at various stages of E and MK differentiation. Furthermore, we investigated the mechanism by which nicotinamide (NIC) promoted terminal MK maturation using an MK‐committed cell line, CHRF‐288‐11 (CHRF). Concomitant with its enhancement of ploidy, NIC strongly enhanced the activity of three TFs with known involvement in terminal MK maturation: FLI‐1, NF‐E2, and p53. Dynamic profiling of TF activity represents a novel tool to complement traditional assays focused on mRNA and protein expression levels to understand progenitor cell differentiation. Biotechnol. Bioeng. 2014;111: 2082–2094.