Sunyoung Joo

Amniotic Fluid-Derived Stem Cells in Regenerative Medicine Research

The stem cells isolated from amniotic fluid present an exciting possible contribution to the field of regenerative medicine and amniotic fluid-derived stem (AFS) cells have significant potential for research and therapeutic applications. AFS cells are multipotent, showing the ability to differentiate into cell types from all three embryonic germ layers. They express both embryonic and adult stem cell markers, expand extensively without feeder cells, double in 36 h, and are not tumorigenic. The AFS cells can be maintained for over 250 population doublings and preserve their telomere length and a normal karyotype. They differentiate easily into specific cell lineages and do not require human embryo tissue for their isolation, thus avoiding the current controversies associated with the use of human embryonic stem (ES) cells. The discovery of the AFS cells has been recent, and a great deal of work remains to be performed on the characterization and use of these cells. This review describes the various differentiated lineages that AFS cells can form and the future of these promising new stem cells in regenerative medicine research.

Engineered multilayer ovarian tissue that secretes sex steroids and peptide hormones in response to gonadotropins.

Although hormone replacement therapy is an option for the loss of ovarian function, hormone delivery through pharmacological means results in various clinical complications. The present study was designed to deliver sex steroids by a functional construct fabricated using encapsulation techniques. Theca and granulosa cells isolated from ovaries of 21-day old rats were encapsulated in multilayer alginate microcapsules to recapitulate the native follicular structure. Cells encapsulated in two other schemes were used as controls to assess the importance of the multilayer structure. The endocrine functions of the encapsulated cells were assessed in vitro for a period of 30 days. Encapsulated cells showed sustained viability during long-term in vitro culture with those encapsulated in multilayer capsules secreting significantly higher and sustained concentrations of 17 β-estradiol (E(2)) than the two other encapsulation schemes (p < 0.05, n = 6) in response to follicle-stimulating hormone (FSH) and luteinizing hormone (LH). In addition, cells in the multilayer microcapsules also secreted activin and inhibin in vitro. In contrast, when granulosa and theca cells were cultured in 2D culture, progesterone (P(4)) secretion increased while E(2) secretion decreased over a 30-day period. In summary, we have designed a multilayer engineered ovarian tissue that secretes sex steroids and peptide hormones and responds to gonadotropins, thus demonstrating the ability to recapitulate native ovarian structure ex vivo.

Syngeneic Myoblast Transplantation Improves Muscle Function in a Murine Model of X-Linked Myotubular Myopathy

X-linked myotubular myopathy (XLMTM) is an isogenic muscle disease characterized by progressive wasting of skeletal muscle, weakness, and premature death of affected male offspring. Recently, the XLMTM gene knock-in mouse, Mtm1 p.R69C, was found to have a similar phenotype as the MTM1 gene mutation in humans (e.g., central nucleation of small myofibers, attenuated muscle strength, and motor unit potentials). Using this rodent model, we investigated whether syngeneic cell therapy could mitigate muscle weakness. Donor skeletal muscle-derived myoblasts were isolated from C57BL6 wild-type (WT) and Mtm1 p.R69C (KI) mice for transplantation into the gastrocnemius muscle of recipient KI mice. Initial experiments demonstrated that donor skeletal muscle-derived myoblasts from WT and KI mice remained in the gastrocnemius muscle of the recipient KI mouse for up to 4 weeks posttransplantation. KI mice receiving syngeneic skeletal muscle-derived myoblasts displayed an increase in skeletal muscle mass, augmented force generation, and increased nerve-evoked skeletal muscle action potential amplitude. Taken together, these results support our hypothesis that syngeneic cell therapy may potentially be used to ameliorate muscle weakness and delay the progression of XLMTM, as application expands to other muscles.

Myogenic-induced mesenchymal stem cells are capable of modulating the immune response by regulatory T cells.

Cell therapy for patients who have intractable muscle disorders may require highly regenerative cells from young, healthy allogeneic donors. Mesenchymal stem cells are currently under clinical investigation because they are known to induce muscle regeneration and believed to be immune privileged, thus making them suitable for allogeneic applications. However, it is unclear whether allogeneic and myogenic-induced mesenchymal stem cells retain their immunomodulatory characteristics. Therefore, our aim was to evaluate the effects of mesenchymal stem cell differentiation on the immune characteristics of cells in vitro. We investigated the immunologic properties of mesenchymal stem cells after myogenic induction. Mesenchymal stem cells were obtained from C57BL/6 mice and the C3H/10T1/2 murine mesenchymal stem cell line. Two different 5-aza-2′-deoxycytidine doses (0.5 and 3 µM) were evaluated for their effects on mesenchymal stem cell skeletal myogenic differentiation potential, immune antigen expression, and mixed lymphocytic reactions. Using a mixed lymphocytic reaction, we determined the optimal splenocyte proliferation inhibition dose. The induction of regulatory T cells was markedly increased by the addition of 3 µM 5-aza-2′-deoxycytidine–treated mesenchymal stem cells. Myogenic-induced mesenchymal stem cells do not elicit alloreactive lymphocyte proliferative responses and are able to modulate immune responses. These findings support the hypothesis that myogenic-induced mesenchymal stem cells may be transplantable across allogeneic barriers.

In vivo transplantation of 3D encapsulated ovarian constructs in rats corrects abnormalities of ovarian failure

Safe clinical hormone replacement (HR) will likely become increasingly important in the growing populations of aged women and cancer patients undergoing treatments that ablate the ovaries. Cell-based HRT (cHRT) is an alternative approach that may allow certain physiological outcomes to be achieved with lower circulating hormone levels than pharmacological means due to participation of cells in the hypothalamus-pituitary-ovary feedback control loop. Here we describe the in vivo performance of 3D bioengineered ovarian constructs that recapitulate native cell–cell interactions between ovarian granulosa and theca cells as an approach to cHRT. The constructs are fabricated using either Ca++ or Sr++ to crosslink alginate. Following implantation in ovariectomized (ovx) rats, the Sr++-cross-linked constructs achieve stable secretion of hormones during 90 days of study. Further, we show these constructs with isogeneic cells to be effective in ameliorating adverse effects of hormone deficiency, including bone health, uterine health, and body composition in this rat model.Cell-based hormone replacement therapy (cHRT) may be an alternative therapy to pharmacological (p)HRT. Here, the authors show that implanted 3D bioengineered ovarian constructs of granulosa and theca cells in ovariectomized rats recapitulate native cell interactions and improve efficacy compared to similar doses of pHRT.

The effect of collagen hydrogel on 3D culture of ovarian follicles.

The in vivo function and phenotype of ovarian follicle cells are determined by many factors. When these cells are removed from the in vivo microenvironment and grown in a 2D in vitro environment, the function of the follicular cells is difficult to preserve. A collagen hydrogel was used to examine the hormone and oocyte maturation of ovary follicles in a 3D culture system. Ovarian follicles from rats were isolated and cultured in various concentration of type I collagen hydrogels ranging from 1% to 7% (weight/volume). Differences in cell survival, follicle growth and development, sex hormone production, and oocyte maturation were seen with the modifications in the collagen hydrogel density and elasticity. The results show the significance of the collagen hydrogel properties on phenotype and function maintenance of the ovarian follicles in a 3D culture system.