Cryopreserved clinical-grade human embryonic stem cell-derived dopaminergic progenitors function in Parkinson's disease models.

AIM: Parkinson's Disease (PD) is a common age-related neurodegenerative disorder with a rising prevalence. Human pluripotent stem cells have emerged as the most promising source of cells for midbrain dopaminergic (mDA) neuron replacement in PD. This study aimed to generate transplantable mDA progenitors for treatment of PD. MATERIALS AND METHODS: Here, we optimized and fine-tuned a differentiation protocol using a combination of small molecules and growth factors to induce mDA progenitors to comply with good manufacturing practice (GMP) guidelines based on our clinical-grade human embryonic stem cell (hESC) line. KEY FINDINGS: The resulting mDA progenitors demonstrated robust differentiation and functional properties in vitro. Moreover, cryopreserved mDA progenitors were transplanted into 6-hydroxydopamine-lesioned rats, leading to functional recovery. SIGNIFICANCE: We demonstrate that our optimized protocol using a clinical hESC line is suitable for generating clinical-grade mDA progenitors and provides the ground work for future translational applications.

A Roadmap for the Production of a GMP-Compatible Cell Bank of Allogeneic Bone Marrow-Derived Clonal Mesenchymal Stromal Cells for Cell Therapy Applications.

BACKGROUND: Allogeneic mesenchymal stromal cells (MSCs) have been used extensively in various clinical trials. Nevertheless, there are concerns about their efficacy, attributed mainly to the heterogeneity of the applied populations. Therefore, producing a consistent population of MSCs is crucial to improve their therapeutic efficacy. This study presents a good manufacturing practice (GMP)-compatible and cost-effective protocol for manufacturing, banking, and lot-release of a homogeneous population of human bone marrow-derived clonal MSCs (cMSCs). METHODS: Here, cMSCs were isolated based on the subfractionation culturing method. Afterward, isolated clones that could reproduce up to passage three were stored as the seed stock. To select proliferative clones, we used an innovative, cost-effective screening strategy based on lengthy serial passaging. Finally, the selected clones re-cultured from the seed stock to establish the following four-tired cell banking system: initial, master, working, and end of product cell banks (ICB, MCB, WCB, and EoPCB). RESULTS: Through a rigorous screening strategy, three clones were selected from a total of 21 clones that were stored during the clonal isolation process. The selected clones met the identity, quality, and safety assessments criteria. The validated clones were stored in the four-tiered cell bank system under GMP conditions, and certificates of analysis were provided for the three-individual ready-to-release batches. Finally, a stability study validated the EoPCB, release, and transport process of the frozen final products. CONCLUSION: Collectively, this study presents a technical and translational overview of a GMP-compatible cMSCs manufacturing technology that could lead to the development of similar products for potential therapeutic applications.

Chicken Interspecies Chimerism Unveils Human Pluripotency.

Human pluripotent stem cells (hPSCs) are commonly kept in a primed state but also able to acquire a more immature naive state under specific conditions in vitro. Acquisition of naive state changes several properties of hPSCs and might affect their contribution to embryonic development in vivo. However, the lack of an appropriate animal test system has made it difficult to assess potential differences for chimera formation between naive and primed hPSCs. Here, we report that the developing chicken embryo is a permissive host for hPSCs, allowing analysis of the pluripotency potential of hPSCs. Transplantation of naive-like and primed hPSCs at matched developmental stages resulted in robust chimerism. Importantly, the ability of naive-like but not of primed hPSCs to form chimera was substantially reduced when injected at non-matched developmental stages. We propose that contribution to chick embryogenesis is an informative and versatile test to identify different pluripotent states of hPSCs.

Isolation, identification and differentiation of human spermatogonial cells on three-dimensional decellularized sheep testis.

Improvement of in vitro culture methods of Spermatogonial Stem Cells (SSCs) is known to be an effective procedure for further study of the process of spermatogenesis and can offer effective therapeutic modality for male infertility. Tissue decellularization by providing natural 3D and extracellular matrix (ECM) conditions for cell growth can be an alternative procedure to enhance in vitro culture conditions. In the present study, the testicular tissues were taken from brain death donors. After enzymatic digestion, the tissue cells were isolated and cultured for four weeks. Then the identity of the SSCs was confirmed using anti-GFRα1 and anti-PLZF antibodies via immunocytochemistry (ICC). The differentiation capacity of SSCs were evaluated by culture of them on a layer of decellularized testicular matrix (DTM) prepared from sheep testis, as well as under two-dimensional (2D) culture with differentiation medium. After four and six weeks of the initiation of differentiation culture, the pre-meiotic, meiotic and post- meiotic genes at the mRNA and protein levels was examined via qPCR and ICC methods, respectively. The results showed that pre-meiotic, meiotic and post-meiotic genes expressions were significantly higher in the cells cultured in DTM substrate (P ≤ 0.01).The present study indicated that, the natural structure of ECM prepare the suitable conditions for further study of the spermatogenesis process in the in vitro and contributes to the maintenance and treatment of male infertility.

Expression analysis of genes encoding TEX11, TEX12, TEX14 and TEX15 in testis tissues of men with non-obstructive azoospermia.

OBJECTIVE: Spermatogenesis is a complex process controlled by a plethora of genes. Changes in expression and function of these genes may thus lead to spermatogenic deficiency and male infertility. TEX11, TEX12, TEX14 and TEX15 are germ cell-specific genes expressed in the testis. TEX11, involved in the initiation and maintenance of chromosome synapses in meiotic chromosomes, has been shown to be essential for meiosis and fertility in males. TEX14, a component of intercellular bridges in germ cells, is required for spermatogenesis and fertility. TEX12 and TEX15 are essential for correct assembly of the synaptonemal complex and thus meiosis progression. METHODS: In order to examine whether changes in expression of these genes is associated with impaired spermatogenesis, expression levels of these genes were quantified by RT-qPCR on samples retrieved from infertile patients submitted to diagnostic testicular biopsy at Royan institute. Samples were divided into two groups of 18 patients with non-obstructive azoospermia considered as case; nine patients with obstructive azoospermia were included in the control group. RESULTS: A significant down-regulation of these genes was observed in the SCOS group when compared to the control group. CONCLUSION: This result suggests that regular expression of TEX11, TEX12, TEX14 and TEX15 is essential for the early stages of spermatogenesis.

Low Focal Adhesion Signaling Promotes Ground State Pluripotency of Mouse Embryonic Stem Cells.

Mouse embryonic stem cells (mESCs) can be maintained in a pluripotent state when cultured with 2 inhibitors (2i) of extracellular signal-regulated kinase (MEK) and glycogen synthase kinase-3 (GSK3), and Royan 2 inhibitors (R2i) of FGF4 and TGFß. The molecular mechanisms that control ESC self-renewal and pluripotency are more important for translating stem cell technologies to clinical applications. In this study, we used the shotgun proteomics technique to compare the proteome of the ground state condition (R2i- and 2i-grown cells) to that of serum. Out of 1749 proteins identified, 171 proteins were differentially expressed (p < 0.05) in the 2i, R2i, and serum samples. Gene ontology (GO) analysis of differentially abundant proteins showed that the focal adhesion signaling pathway significantly down-regulated under ground state conditions. mESCs had highly adhesive attachment under the serum condition, whereas in the 2i and R2i culture conditions, a loss of adhesion was observed and the cells were rounded and grew in compact colonies on gelatin. Quantitative RT-PCR showed reduced expression of the integrins family in the 2i and R2i conditions. The serum culture had more prominent phosphorylation of focal adhesion kinase (FAK) compared to 2i and R2i cultures. Activity of the extracellular signal-regulated kinase (ERK)1/2 decreased in the 2i and R2i cultures compared to serum. Activation of integrins by Mn2+ in the 2i and R2i cultures resulted in reduced Nanog and increased the expression of lineage marker genes. In this study, we demonstrated that reduced focal adhesion enabled mESCs to be maintained in an undifferentiated and pluripotent state.