Modeling autosomal dominant optic atrophy using induced pluripotent stem cells and identifying potential therapeutic targets

OPA1 +/− -iPSCs (OL) are unfavorable to differentiate into neural rosette. Culture conditions were the same as those in Fig. 3. Panel A shows day 5 EBs, which were derived from control and OL-iPSCs, respectively. Morphologically, OL-EBs look similar to control EBs. Panel B shows neuron rosettes (NRs) after 5 days of EB attachment. Fewer and smaller neuron rosettes were derived from OL-EBs, compared with neuron rosettes from control EBs. Figure S2 OPA1 +/− -iPSCs (OL) failed to differentiate into RGCs with culture medium supplemented with 10 % FBS and DAPT. Differentiation method in Fig. 4 was followed. Putative RGCs were fixed on day 24 before fixation, followed by IF staining. Upper panel shows staining of TUJ1 (green) and BRN3a (red), while lower panel displays staining of TUJ1 (green) and ISLET1 (red). The scale bars equal 50 μM. Figure S3 IF analysis of RGCs derived from the control and VO-iPSCs on day 38. Neurospheres were plated onto PLO/L-coated plates and cultured in hESC medium containing 10 μM DAPT and 10 % FBS for 21 days before fixation with 4 % PFA on day 38. Cells were stained with TUJ1 (green), BRN3a (red) and ISLET-1 (red) antibodies. The scale bars equal 20 μM. Figure S4 NIM promoted OPA1 +/− -RGC (OL) generation detected by IF. Putative OL-RGCs derived from OL-iPSCs were cultured with hESC medium containing 10 μM DAPT, 10 % FBS, and 10 % neural induction medium (NIM) for 14 days before fixation on day 31. Upper panel shows TUJ1 (green) and BRN3a (red) staining. Lower panel shows staining of TUJ1 (green) and ISLET1 (red). The scale bars equal 50 μM. Figure S5 Quantification of RGC differentiation efficiency. The culture medium used for RGC differentiation contained 10 % neural induction medium. Samples were normalized to the control BRN3a/DAPI staining or the control ISLET-1/DAPI staining. The number of BRN3a or ISLET-1 signals versus the number of DAPI signals was calculated. A p-value of 0.084 was obtained for the BRN3a signal comparison between the control and VO, and a p-value of 0.076 was obtained for the ISLET-1 signal comparison between the control and VO. Student’s t-test was used to analyze differences between two groups. Figure S6 Noggin rescued OPA1 +/− -iPSC (OL) differentiation into RGCs confirmed by IF. Mature EBs were cultured with hES medium containing 10 % FBS and 100 ng/mL Noggin to obtain neuron rosettes, followed by culturing putative RGCs with hES medium containing 10 % FBS, DAPT and 100 ng/mL Noggin. Cells were fixed with 4 % PFA on day 31. Upper panel shows IF staining of TUJ-1 (green) and BRN3a (red), whereas lower panel shows staining of TUJ-1 (green) and ISLET1 (red). The scale bars equal 50 μM in both panels. Figure S7 Addition of 17β-estradiol in RGC differentiation medium promoted generation of OPA1 +/− -RGCs (OL). Putative RGCs were incubated with 100 nM 17β-estradiol in the cell culture medium during all of the differentiation stages, followed by fixation on day 31. Upper panel shows IF staining against TUJ1 (green) and BRN3a (red). Lower panel shows TUJ1 (green) and ISLET-1 (red) staining. The scale bars equal 50 μM. (PPTX 12904 kb

From Blood to the Brain: Can Systemically Transplanted Mesenchymal Stem Cells Cross the Blood-Brain Barrier?

Systemically infused mesenchymal stem cells (MSCs) are emerging therapeutics for treating stroke, acute injuries, and inflammatory diseases of the central nervous system (CNS), as well as brain tumors due to their regenerative capacity and ability to secrete trophic, immune modulatory, or other engineered therapeutic factors. It is hypothesized that transplanted MSCs home to and engraft at ischemic and injured sites in the brain in order to exert their therapeutic effects. However, whether MSCs possess the ability to migrate across the blood-brain barrier (BBB) that separates the blood from the brain remains unresolved. This review analyzes recent advances in this area in an attempt to elucidate whether systemically infused MSCs are able to actively transmigrate across the CNS endothelium, particularly under conditions of injury or stroke. Understanding the fate of transplanted MSCs and their CNS trafficking mechanisms will facilitate the development of more effective stem-cell-based therapeutics and drug delivery systems to treat neurological diseases and brain tumors

Chemically Induced Specification of Retinal Ganglion Cells From Human Embryonic and Induced Pluripotent Stem Cells

The loss of retinal ganglion cells (RGCs) is the primary pathological change for many retinal degenerative diseases. Although there is currently no effective treatment for this group of diseases, cell transplantation to replace lost RGCs holds great potential. However, for the development of cell replacement therapy, better understanding of the molecular details involved in differentiating stem cells into RGCs is essential. In this study, a novel, stepwise chemical protocol is described for the differentiation of human embryonic stem cells and induced pluripotent stem cells into functional RGCs. Briefly, stem cells were differentiated into neural rosettes, which were then cultured with the Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). The expression of neural and RGC markers (BRN3A, BRN3B, ATOH7/Math5, γ-synuclein, Islet-1, and THY-1) was examined. Approximately 30% of the cell population obtained expressed the neuronal marker TUJ1 as well the RGC markers. Moreover, the differentiated RGCs generated action potentials and exhibited both spontaneous and evoked excitatory postsynaptic currents, indicating that functional and mature RGCs were generated. In combination, these data demonstrate that a single chemical (DAPT) can induce PAX6/RX-positive stem cells to undergo differentiation into functional RGCs

Chemically induced specification of retinal ganglion cells from human embryonic and induced pluripotent stem cells.

The loss of retinal ganglion cells (RGCs) is the primary pathological change for many retinal degenerative diseases. Although there is currently no effective treatment for this group of diseases, cell transplantation to replace lost RGCs holds great potential. However, for the development of cell replacement therapy, better understanding of the molecular details involved in differentiating stem cells into RGCs is essential. In this study, a novel, stepwise chemical protocol is described for the differentiation of human embryonic stem cells and induced pluripotent stem cells into functional RGCs. Briefly, stem cells were differentiated into neural rosettes, which were then cultured with the Notch inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT). The expression of neural and RGC markers (BRN3A, BRN3B, ATOH7/Math5, γ-synuclein, Islet-1, and THY-1) was examined. Approximately 30% of the cell population obtained expressed the neuronal marker TUJ1 as well the RGC markers. Moreover, the differentiated RGCs generated action potentials and exhibited both spontaneous and evoked excitatory postsynaptic currents, indicating that functional and mature RGCs were generated. In combination, these data demonstrate that a single chemical (DAPT) can induce PAX6/RX-positive stem cells to undergo differentiation into functional RGCs

From blood to the brain: can systemically transplanted mesenchymal stem cells cross the blood-brain barrier?

Systemically infused mesenchymal stem cells (MSCs) are emerging therapeutics for treating stroke, acute injuries, and inflammatory diseases of the central nervous system (CNS), as well as brain tumors due to their regenerative capacity and ability to secrete trophic, immune modulatory, or other engineered therapeutic factors. It is hypothesized that transplanted MSCs home to and engraft at ischemic and injured sites in the brain in order to exert their therapeutic effects. However, whether MSCs possess the ability to migrate across the blood-brain barrier (BBB) that separates the blood from the brain remains unresolved. This review analyzes recent advances in this area in an attempt to elucidate whether systemically infused MSCs are able to actively transmigrate across the CNS endothelium, particularly under conditions of injury or stroke. Understanding the fate of transplanted MSCs and their CNS trafficking mechanisms will facilitate the development of more effective stem-cell-based therapeutics and drug delivery systems to treat neurological diseases and brain tumors

From blood to the brain: can systemically transplanted mesenchymal stem cells cross the blood-brain barrier?

Systemically infused mesenchymal stem cells (MSCs) are emerging therapeutics for treating stroke, acute injuries, and inflammatory diseases of the central nervous system (CNS), as well as brain tumors due to their regenerative capacity and ability to secrete trophic, immune modulatory, or other engineered therapeutic factors. It is hypothesized that transplanted MSCs home to and engraft at ischemic and injured sites in the brain in order to exert their therapeutic effects. However, whether MSCs possess the ability to migrate across the blood-brain barrier (BBB) that separates the blood from the brain remains unresolved. This review analyzes recent advances in this area in an attempt to elucidate whether systemically infused MSCs are able to actively transmigrate across the CNS endothelium, particularly under conditions of injury or stroke. Understanding the fate of transplanted MSCs and their CNS trafficking mechanisms will facilitate the development of more effective stem-cell-based therapeutics and drug delivery systems to treat neurological diseases and brain tumors

From blood to the brain: can systemically transplanted mesenchymal stem cells cross the blood-brain barrier?

Systemically infused mesenchymal stem cells (MSCs) are emerging therapeutics for treating stroke, acute injuries, and inflammatory diseases of the central nervous system (CNS), as well as brain tumors due to their regenerative capacity and ability to secrete trophic, immune modulatory, or other engineered therapeutic factors. It is hypothesized that transplanted MSCs home to and engraft at ischemic and injured sites in the brain in order to exert their therapeutic effects. However, whether MSCs possess the ability to migrate across the blood-brain barrier (BBB) that separates the blood from the brain remains unresolved. This review analyzes recent advances in this area in an attempt to elucidate whether systemically infused MSCs are able to actively transmigrate across the CNS endothelium, particularly under conditions of injury or stroke. Understanding the fate of transplanted MSCs and their CNS trafficking mechanisms will facilitate the development of more effective stem-cell-based therapeutics and drug delivery systems to treat neurological diseases and brain tumors

Facile supermolecular aptamer inhibitors of L-selectin.

Multivalent interactions occur frequently in nature, where they mediate high-affinity interactions between cells, proteins, or molecules. Here, we report on a method to generate multivalent aptamers (Multi-Aptamers) that target L-selectin function using rolling circle amplification (RCA). We find that the L-selectin Multi-Aptamers have increased affinity compared to the monovalent aptamer, are specific to L-selectin, and are capable of inhibiting interactions with endogenous ligands. In addition, the Multi-Aptamers efficiently inhibit L-selectin mediated dynamic adhesion in vitro and homing to secondary lymphoid tissues in vivo. Importantly, our method of generating multivalent materials using RCA avoids many of the challenges associated with current multivalent materials in that the Multi-Aptamers are high affinity, easily produced and modified, and biocompatible. We anticipate that the Multi-Aptamers can serve as a platform technology to modulate diverse cellular processes

Phenotypic and functional characterization of Bst+/− mouse retina

The belly spot and tail (Bst+/−) mouse phenotype is caused by mutations of the ribosomal protein L24 (Rpl24). Among various phenotypes in Bst+/− mice, the most interesting are its retinal abnormalities, consisting of delayed closure of choroid fissures, decreased ganglion cells and subretinal vascularization. We further characterized the Bst+/− mouse and investigated the underlying molecular mechanisms to assess the feasibility of using this strain as a model for stem cell therapy of retinal degenerative diseases due to retinal ganglion cell (RGC) loss. We found that, although RGCs are significantly reduced in retinal ganglion cell layer in Bst+/− mouse, melanopsin+ RGCs, also called ipRGCs, appear to be unchanged. Pupillary light reflex was completely absent in Bst+/− mice but they had a normal circadian rhythm. In order to examine the pathological abnormalities in Bst+/− mice, we performed electron microscopy in RGC and found that mitochondria morphology was deformed, having irregular borders and lacking cristae. The complex activities of the mitochondrial electron transport chain were significantly decreased. Finally, for subretinal vascularization, we also found that angiogenesis is delayed in Bst+/− associated with delayed hyaloid regression. Characterization of Bst+/− retina suggests that the Bst+/− mouse strain could be a useful murine model. It might be used to explore further the pathogenesis and strategy of treatment of retinal degenerative diseases by employing stem cell technology