Patent Application
20150125432
The field of the invention relates to cell therapy.
Inner retinal degenerative diseases, such as selective and progressive loss of retinal ganglion cells (RGCs), pose a major threat to vision. Currently there is no effective cure or treatment to reverse the loss of RGCs. Transplantation of stem or progenitor cells may have great therapeutic potential for treatment of neurodegenerative diseases in general by providing therapeutic benefits through both neuroprotective and cell replacement mechanisms and some regenerative potential of cell transplantation has already been shown for the outer retina. However, it has proven difficult to achieve similar success for replacement of RGCs, as they adopt highly specialized properties and form numerous synaptic connections with other neurons. Due to the inhibitory environment present in the adult neural retina and lineage restriction of engrafted cells, limited integration and RGC-specific differentiation of engrafted cells has been found in the inner retina when cells have been intravitreally transplanted into the uninjured eye.
The invention provides the use of human persistent fetal vasculature neural progenitor cells for transplantation. These purified cells integrate into the retinal ganglion cell layer of the eye after transplantation into the eye, e.g., into the vitreous of the eye, thereby overcoming problems and/or drawbacks of earlier approaches to treat such degenerative diseases. For example, a cell-based method of therapy is carried out by providing a purified population of human persistent fetal vasculature neural progenitor cells (hNPPFV) and transplanting the cells into an ocular tissue of a recipient subject. The methods are suitable for not only humans but other animals as well, e.g., companion animals such as dogs, cats, and the like as well as livestock or other animals. The cells are obtained from the individual to be treated or a member of the same species. In one example, the cells are transplanted into an inner retina location of an eye. In some cases, the cells have been modified to increase expression of insulin-like growth factor-1 (IGF-1) or insulin-like growth factor-binding protein (IGFBP)-1. For example, the cells to be transplanted have been transfected with a nucleic acid encoding IGF-1.
Thus, the invention encompasses a human persistent fetal vascular tissue cell or cell line comprising a neuronal progenitor marker such as nestin, Pax6, or Ki67. For example, the marker(s) are expressed on the surface or in the cytoplasm of the cell. In some embodiments, the cell further comprises a retinal neuronal marker such as β-III-tubulin or Brn3a. The cells comprise a neural morphological phenotype or express a mature neuronal marker in the presence of a neural phenotype induction factor such as a an environmental cue, e.g., contact with a neural cell differentiation factor. Examples of such phenotype induction factors or neural cell differentiation factors include small molecules such as retinoic acid or neurotrophins or neurotrophic factors. Neurotrophins are a family of proteins that induce the development, survival, and/or function of neurons.
The term neurotrophin may be used as a synonym for neurotrophic factor, but the term neurotrophin is more generally reserved for four structurally related factors: nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neurotrophin-4 (NT-4), with neurotrophic factor additionally referring to the GDNF family of ligands and ciliary neurotrophic factor (CNTF). Exemplary mature neuronal marker include β-III-tubulin, synaptophysin, or NF200.
Also within the invention is a method of promoting survival or axonal outgrowth of a retinal ganglion cell (RGC), comprising contacting the RGC with the population of persistent fetal vasculature neural progenitor cells described above. A method of conferring neuroprotection to a retinal ganglion cell in a subject is also within the invention. Neuroprotection is conferred by administering to an ocular tissue comprising a RGC a purified IGF-1 or a purified cell expressing an increased level of IGF-1. Preferably, the cell secretes IGF-1. For example, the cell comprises a vector containing a coding sequence encoding human IGF-1 or a neuroprotective fragment thereof.
The candidate subject to be treated has been diagnosed with a degenerative disease of an eye. For example, the degenerative disease comprises glaucoma, ischemic optic neuropathy, optic neuritis, or inherited mitochondrial optic neuropathies. The methods are suitable for any subject that has been characterized as suffering from or at risk of developing a neurodegenerative disease of an eye, e.g., degenerative disease or disorder of the inner retina.
A purified population of human persistent fetal vasculature neural progenitor cells comprising an heterologous or exogenous nucleic acid encoding a neuroprotective polypeptide such as an exogenous IGF-1 encoding nucleic acid or expressing/secreting a neuroprotective IGF-1 polypeptide is also within the invention. The IGF-1 comprises the full mature protein or a fragment that possesses a neuroprotective activity of the full-length protein. An exemplary human IGF-1 protein is described in GenBank: CAA01955.1, CAA01954.1 (GI:1247519), or P05019 (IGF1_HUMAN, showing molecule processing, regions/domains, and disulfide bond locations; last modified Apr. 18, 2012).
For example, IGF-1 precursor protein comprises amino acid residue 1-119, and the mature peptide comprises amino acid residues 15-84 of the sequence shown below. Fragments include any peptide that is less than the full length IGF-1, e.g., a fragment is less than 119 residues or less than the 69 amino acids, e.g., an IGF-1 fragment comprises 10, 20, 25, 30, 40, 50, 60, 65, 70, 75, 80, 90, 100, 110, 115 amino acids. IGF-1 activity of fragments is tested using known methods.
1 malclltfts satagpetlc gaelvdalqf vcgdrgfyfn kptgygsssr rapqtgivde
61 ccfrscdlrr lemycaplkp aksarsvraq rhtdmpktqk evhlknasrg sagnknyrm (SEQ ID NO: 25)
Exemplary IGF-1 encoding nucleic acid sequences include NM—202494.2, NM—202470.2, NM—202468.2, NM—005716.3, or NM—202469.2. For example, such a population of cells is intravitreally transplanted into a subject to confer a clinical benefit.
Also within the invention is the use of a persistent fetal vasculature neural progenitor cell for drug discovery, wherein the cell comprises a nucleic acid encoding a reporter gene. For example, screening assays, e.g., high throughput screening assays, are carried out by contacting the cells expressing a reporter gene (e.g., encoding a detectable gene product such as a green fluorescent protein (GFP), TDtomato, or any number of other reporter gene products known in the art) with a test compound, e.g., a candidate drug, and measuring level of expression of the reporter gene (transcript or gene product) as a read-out for a specific cellular activity. Alternatively, the cells are interrogated by employing a “live-dead” reporter assay.
All polynucleotides and polypeptides of the invention are purified and/or isolated. As used herein, an “isolated” or “purified” nucleotide or polypeptide is substantially free of other nucleotides and polypeptides. Purified nucleotides and polypeptides are also free of cellular material or other chemicals when chemically synthesized. Purified compounds are at least 60% by weight (dry weight) the compound of interest. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight the compound of interest. For example, a purified nucleotides and polypeptides is one that is at least 90%, 91%, 92%, 93%, 94%, 95%, 98%, 99%, or 100% (w/w) of the desired nucleic acid or polypeptide by weight.
Purity is measured by any appropriate standard method, for example, by column chromatography, thin layer chromatography, or high-performance liquid chromatography (HPLC) analysis. The nucleotides and polypeptides are purified and used in a number of products for consumption by humans as well as animals, such as companion animals (dogs, cats) as well as livestock (bovine, equine, ovine, caprine, or porcine animals, as well as poultry). A purified or isolated polynucleotide (ribonucleic acid (RNA) or deoxyribonucleic acid (DNA)) is free of the genes or sequences that flank it in its naturally-occurring state. For example, the DNA is a cDNA. “Purified” also defines a degree of sterility that is safe for administration to a human subject, e.g., lacking infectious or toxic agents.
Heterologous DNA or heterologous nucleic acid refers to a DNA molecule or a nucleic acid, or a population of DNA molecules or a population of nucleic acids, that do not exist naturally within a given host cell. DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e. endogenous DNA) so long as that host DNA is combined with non-host DNA (i.e. exogenous DNA). For example, a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a promoter is considered to be a heterologous DNA molecule. Conversely, heterologous DNA can comprise an endogenous structural gene operably linked with an exogenous promoter. A heterologous protein is a protein that is expressed by the host cell and encoded by the heterologous. nucleic acid.
Publications, U.S. patents and applications, Genbank/NCBI accession numbers, and all other references cited herein, are hereby incorporated by reference.
Persistent fetal vasculature (PFV) is a potentially serious developmental anomaly in human eyes, which results from a failure of the primary vitreous and the hyaloid vascular systems to regress during development. Fibrovascular membranes harvested from subjects with PFV contain neural progenitor cells (NPPFV cells). A neural progenitor cell type was isolated from human persistent fetal vascular tissue. In this condition, the hyaloidal vascular system that nourishes the developing lens fails to regress, leaving a whitish membrane in the anterior vitreous behind the lens. This tissue contains neural progenitor cells. Studies were therefore carried out to examine whether these NPPFV cells exhibit characteristics of neuronal progenitor cells (such as antigenic and genetic profiles) and whether they were capable of differentiating into retinal neurons. To explore whether NPPFV cells have potential for cell-based inner retinal transplantation therapy, NPPFV cells were intravitreally transplanted into adult C57BL/6 mice and their integration and differentiation in the inner retina examined.
NPPFV cells highly express neuronal progenitor markers [nestin (e.g., NCBI Reference Sequence: NP—006608.1; GENBANK Accession NM—006617.1), Pax6 (e.g., GENBANK Accession AAX56950.1), and/or Ki67 (e.g., GENBANK CAA46520.1, NP—001139438.1., NM—001145966.1., NP—002408.3. NM—002417.4.)] as well as retinal neuronal markers [β-III-tubulin (e.g., GENBANK NP—001184110.1., NM—001197181.1., NP—006077.2. NM—006086.3.) and/or Brn3a (e.g., GENBANK NP—006228.3., NM—006237.3.)]. In the presence of retinoic acid and neurotrophins, these cells acquire a neural morphological appearance in vitro, including a round soma and extensive neurites, and express mature neuronal markers [β-III-tubulin and/or NF200 (e.g., NP—066554.2., NM—021076.3.)]. Further experiments, including real-time qRT-PCR to quantify characteristic gene expression profiles of these cells and Ca2+ imaging to evaluate the response to stimulation with different neurotransmitters, indicated that NPPFV cells resemble a more advanced stage of retinal development and show more differentiation toward inner retinal neurons rather than photoreceptors. To explore the potential of inner retinal transplantation, NPPFV cells were transplanted intravitreally into eyes of adult C57BL/6 mice. Engrafted NPPFV cells survived well in the intraocular environment in presence of an immunosuppressant such as rapamycin, and some cells migrated into the inner nuclear layer of the retina one week post-transplantation. Three weeks after transplantation, NPPFV cells were observed to some migrate and integrate in the inner retina. In response to daily intraperitoneal injections of retinoic acid, a portion of transplanted NPPFV cells exhibited retinal ganglion cell-like morphology and expressed mature neuronal markers [β-III-tubulin and synaptophysin (e.g., GENBANK NP—003170.1., NM—003179.2.)]. These findings indicate that fibrovascular membranes from human PFV harbor a population of neuronal progenitors are useful for cell-based therapy for degenerative diseases of the inner retina.
The following materials and methods were used to carry out the data described herein.
Isolation of Primary Cells and PFV Cell Culture.
PFV membranes (
Cells then were examined for morphological features, antigenic profiles, gene expression and neurotransmitter receptors.
Immunocytochemical Examination.
NPPFV cells were seeded on slide-chambers (VWR, Batavia, Ill., USA) and cultured in a differentiation-conditioned medium for at least 7 days or in normal growth medium for examining their antigenic profiles. Cells were fixed for 10 min in 4% buffered paraformaldehyde, washed in PBS and blocked with 10% goat serum (Vector, Burlingame, Calif., USA)/PBS solution for 30 min before immunocytochemical staining. Primary antibodies [including β-III-tubulin (Sigma), nestin (Sigma), Pax6 (Chemicon-Millipore, Billerica, Mass., USA), Ki67 (Vector), Brn3a (Chemicon), Brdu (ABCAM, Cambridge, Mass., USA), NF200 (neurofilament-200, Sigma), recoverin (Chemicon), RPE-65 (Chemicon), CRALBP (ABCAM), GFAP (Sigma), α-SMA (ABCAM), FSP1 (Sigma), CD31 (ABCAM) were diluted in 2% goat serum/PBS solution to appropriate concentrations for incubating cells at 4° C. overnight. Cells were rinsed with PBS and incubated with a secondary antibody consisting of either Cy3 (Chemicon 1:500) or FITC (Chemicon 1:300) at room temperature for 30 min on the following day. After rinsing in PBS, slides were mounted with Vectashield mounting medium containing DAPI (Vector) and visualized under an inverted fluorescence microscope (Olympus 1X51). All immunocytochemical analyses were repeated 3 or more times from the cells at passage 2 to 5. Related isotype immunoglobulins were used as negative controls (Chemicon).
Evaluation of Gene Expression by Real-Time aRT-PCR.
To determine the gene profiles of NPPFV cells, real-time qRT-PCR was performed to compare designated mRNA expression of NPPFV cells with those of human retinal progenitor cells (hRPCs). hRPCs have been isolated from human fetal tissue and well characterized in vitro. The isolation procedures for those cells are known (Klassen, H. J.; Ziaeian, B.; Kirov, I. I.; Young, M. J.; Schwartz, P. H. Isolation of retinal progenitor cells from post-mortem human tissue and comparison with autologous brain progenitors. J. Neurosci. Res. 77:334-343; 2004). Retinas of donors with an estimated age of 18 weeks of gestation were cut into small pieces on a dry petri dish under a tissue culture hood and enzymatically digested in a sterile container at 37° C. with periodic removal of supernatants and refilling with fresh digestion solution. Harvested cells from the supernatants after centrifugation were resuspended in cell-free retinal progenitor-conditioned medium, and then cells were transferred to fibronectin-coated tissue culture flasks containing fresh media. In the present study, both cell types (NPPFV and RPC) from passage 0 were cultured in the same medium after the original isolation from tissue samples to eliminate any possible influence from different culture conditions on their respective gene expressions. The mRNA expression of various transcription factors and retinal specific proteins has been detected by PCR in early passages of cultured hRPCs. Therefore, hRPCs can serve as a well-established control for NPPFV cells in real-time-qPCR experiments. Total RNA was extracted from NPPFV cells and hRPCs (both at passage 5) using an RNA isolation kit (Qiagen, RNeasy Mini Kit). To ensure samples without genomic DNA contamination, total RNA was treated with DNase (Qiagen, RNase-Free DNase Set) and cDNA was synthesized using a Synthesis Kit (Bio-rad). Total cDNA (1 μl) was loaded in each well, mixed with PCR master mix (TaqMan Universal, Applied Biosystems, Foster City, Calif.) and pre-designed primers (IDT, San Diego, Calif.) for Pax6, nestin, ATOH7, recoverin, rhodopsin, SNAP25, STXBP1, RAPSN and THY1, respectively (Listed in Table 1). The procedure for real-time qRT-PCR included 2 min at 50° C., 15 min at 95° C., followed by 40 cycles of 15 s at 95° C., 30 s at 55° C., and 30 s at 72° C. (ABI PRISM 7900 HT; Applied Biosystems). Expression (evaluated as fold change for each target gene) was normalized to glyceraldehyde-3-phosphate dehydrogenase (a housekeeping gene) in hRPCs following the well-established delta-delta method (Schmittgen, T. D.; Livak, K. J. Analyzing real-time PCR data by the comparative C(T) method. Nat. Protoc. 3(6):1101-1108; 2008.). All assays were performed in triplicate. In addition, a non-template control was included in the experiment to estimate DNA contamination of isolated RNA and reagents.
Estimation of Intracellular Ca2+ in NPPFV Cells.
To investigate the neurotransmitter receptors expression on NPPFV cells, Ca2+ imaging was performed by loading cells with the ratiometric Ca2+ sensitive Fura-2 dye. Cells were incubated at 37° C. for 30 min in X-vivo medium (containing 3% FBS, 5 μM Fura-2 tetra-acetoxymethyl ester, 8 μM pluronic acid F127 and 250 μM sulfinpyrazone). Cells were washed in modified Mg2+-free Hank's balanced salt solution (HBSS, 2.6 mM CaCl2, 15 mM HEPES [pH 7.4] and 250 μM sulfinpyrazone). Five types of neurotransmitters, γ-aminobutyric acid (GABA), glutamate (Glu), glycine (Gly), dopamine (Dopa) and acetylcholine (Ach) were dissolved in modified HBSS at a concentration of 0.1 mM. The largest dynamic range for Ca2+-dependent fluorescence signals is obtained by excitation at 340 nm and 380 nm, and detecting their ratio of emission fluorescence intensities at around 510 nm. From this ratio, the concentration of intracellular Ca2+ ([Ca2+]i) can be estimated, using dissociation constants that are derived from calibration curves. By using the ratio of fluorescence intensities produced by excitation at two wave lengths, factors such as uneven dye distribution and photo bleaching are minimized (InCyt Im2TM Ratio Imaging System, Cincinnati, Ohio). The change of [Ca2+]i was calculated from the difference between the peak Ca2+ concentration evoked by each agonist and the control value of HBSS (before the addition of agonist). At least 80 cells were selected for analyzing the change of [Ca2+]i evoked by each agonist, averaged over three repetitions.
Inner Retinal Transplantation.
For studying their potential for inner retinal transplantation, NPPFV cells or hRPCs were intravitreally injected to 20 and 10 C57BL/6 mice, respectively. Animals were maintained in standard animal facility. To trace the transplanted cells, NPPFV cells and hRPCs were infected with an AAV2 or retrovirus vector harboring EGFP (HGTI, Boston, Mass.) following the instructions of each transfection kit, respectively. Mice (4-6 weeks of age) were deeply anesthetized with an intraperitoneal injection of ketamine (120 mg/kg) and xylazine (20 mg/kg) and pupils were dilated with 0.5% topical tropicamide. Dissociated GFP-positive cells (1×105 cells/2 μl) were suspended in HBSS were vitreous cavity of the eye through a glass micropipette connected to a 10 μl Hamilton syringe via polyethylene tubing. Sham-injected mice received HBSS without cells. All experimental animals received injection in one eye and the other eye was used as an untreated control. A small puncture in the cornea (paracenthesis) was used to reduce the intraocular pressure during the transplantation surgery. Rapamycin (2 mg/kg·day) (LC laboratories, Woburn, USA) was administered to all surgical animals to ensure the survival of the xenograft. To facilitate the differentiation of engrafted NPPFV cells, 10 mice were given intraperitoneal injections of retinoic acid (RA, 2 mg/kg·day, Sigma) starting one day prior to transplantation and continuing until termination of the experiment. Of the 10 mice transplanted with hRPCs, 5 received Rapamycin, another 5 received Rapamycin and RA. Animals received terminal anesthesia on week 1 or week 3 after transplantation and the eyes were harvested after intracardial perfusion with 4% paraformaldehyde in PBS. Eyes were cryosectioned at 10 μm and examined under a Lecis TSC SP5 confocal microscope to evaluate the expression and location of different markers. Anti-GFP antibody (1:100, ABCAM, USA) was used to enhance the fluorescence of prelabled-GFP, while anti-β-III-tubulin (1:500, Sigma, USA), anti-GFAP (1:300, Sigma, USA) and anti-synaptophysin (1:100, DAKO, USA) antibodies helped to assess the differentiation of the transplanted cells. To estimate the survival and migration of grafted cells, serial sections on 4 randomly selected eyes that had undergone NPPFV transplantation were performed. Every 10th serial section was counted, measuring 10 μM in thickness as to avoid redundancy of cell counts.
Statistical Analysis.
Statistical analysis was performed using the SPSS 12.0 software package. Results are expressed as mean±SEM (standard error of mean).
Differences between groups were compared by using One-Way ANOVA followed up with Tukey's test or t test, as appropriate, and two-tailed p values are reported.
hNPPFV Progenitors for Retinal Transplantation
Immunocytochemical characterization of NPPFV cells was carried out as described above. Following surgical dissection of clinical PFV membranes (funduscope of PFV subject,
Further incorporation of Brdu and β-III-tubulin staining revealed a subpopulation of proliferating NPPFV cells that were smaller in size with fewer projections than their non-dividing counterparts in the same batch (
Gene expression profiles of NPPFV cells were evaluated. Characteristic markers of retinal progenitors (Pax6, nestin and ATOH7), photoreceptors (recoverin and rhodopsin), synapse-related proteins (SNAP25, STXBP1 ad RAPSN) and retinal ganglion cell (THY1) were selected to establish the gene expression profile of NPPFV cells. Real-time qRT-PCR revealed lower expression of retinal progenitor and photoreceptor markers in NPPFV cells than in hRPCs (t test, all p<0.05) (
Relative levels of intracellular calcium were determined. The relative number of undifferentiated and differentiated NPPFV cells was determined, and their Ca2+ concentration was tested using Fura-2 dye after stimulation with different neurotransmitters. A very small portion of undifferentiated and differentiated NPPFV cells responded to Dopa (6.41±7.14% vs. 7.89±9.18%), Ach (10.96±8.12% vs. 8.23±5.41%), GABA (14.94±11.73% vs. 12.99±4.86%) and Gly (16.67±10.62% vs. 21.05±9.72%) and no significant difference was found in each group (t test, all p>0.05). However, Glu stimulation elicited a large response in undifferentiated and differentiated NPPFV cells (48.98±11.67% vs. 87.88±4.97%) compared to other transmitters (One-Way ANOVA, p<0.01). In addition, Glu evoked a response in a higher proportion of differentiated NPPFV cells than undifferentiated cells (t test, p<0.01) (
Some cells that responded to neurotransmitter stimulation were selected for measurements of relative [Ca2++]i (indicated by white arrows in
Survival and migration of transplanted NPPFV cells and hRPCs was studied.
Transplanted NPPFV cells were tracked in vivo by their expression of EGFP, which was enhanced by anti-GFP antibody staining. DAPI was used to identify the nuclei of live cells. Generally, engrafted cells pooled together in the posterior vitreous and remained as discrete clumps adjacent to the ganglion cell layer (GCL) at week 1 post-transplantation (
Some migratory NPPFV cells exhibited characteristically neural cell morphology, including long and slender projections. Although some migration of engrafted NPPFV cells was found in host retina, the anatomic structure of the retina appeared morphologically normal with distinct structural layers as revealed by DAPI labeling (
Integration and differentiation of transplanted NPPFV cells was evaluated. Typical markers of glial cells and mature neurons were applied for checking the neuronal behaviors of the NPPFV cells in the host retina. Immunolocalization of β-III-tubulin (red fluorescence) expression of engrafted cells indicated that a small number of the engrafted cells had already merged in the GCL at 3 week post-transplantation (
Post-transplanted differentiation into mature retinal neurons or glial cells was not clearly observed in the engrafted cells. Induction of the differentiation of the transplanted cells was attempted by modulating the retinal microenvironment with intraperitoneal treatment of RA. After 3 weeks of treatment, β-III-tubulin was detected in these GFP-positive engrafts that naturally integrated into the GCL and inner nuclear layer of the host retina (white arrows,
If any possible therapeutic effects of replacing lost RGCs with transplanted stem cells are to be achieved for degenerative inner retinal conditions, it is essential that a suitable cell type be chosen for transplantation and a more bio-compatible method be used to facilitate the integration and differentiation of engrafted cells in the host tissue. Glaucoma is not be the only inner retinal condition benefits from restoration of RGCs. RGC protection and/or replacement is applicable to other diseases that result in the death or dysfunction of RGCs, such as ischemic optic neuropathy, optic neuritis, and inherited mitochondrial optic neuropathies.
Patient-derived or donor neural progenitor cell type isolated from human persistent fetal vascular tissue provide a solution to cell therapy problems of previous approaches. In PFV, the hyaloidal vascular system that nourishes the developing lens fails to regress, leaving a whitish membrane in the anterior vitreous behind the lens. As described herein, this tissue contains neural progenitor cells. NPPFV cells exhibited characteristics of neuronal progenitor cells (such as antigenic and genetic profiles) and were found to be capable of differentiating into retinal neurons.
The findings from immunohistochemical examination, real-time qRT-PCR and Ca2+ imaging confirm that NPPFV cells exhibit characteristics of neuronal progenitor cells and were induced to differentiate into mature neurons in vitro. As an appropriate source of stem cells is fundamental to transplantation therapy, NPPFV cells can be easily grown from the surgically dissected tissue from human subjects with PFV, cryopreserved, and passaged for relatively long periods of time, e.g., 10, 20, 25, 50, 100 or more passages. This suggests that these cells are useful for clinical application. Intravitreal transplantation of NPPFV cells demonstrated that they survive well and migrate into the inner retina. Furthermore, modulation of the retinal microenvironment by intraperitoneal injection of RA significantly increases the differentiation of engrafts into retinal ganglion-like mature neurons, which highlights the utility of facilitating engraft integration by modulating the in vivo microenvironment. These results indicate that neural progenitor cells derived from the PFV membrane are well suited for cell-based therapy for inner retinal neurodegenerative diseases, such as glaucoma and that the therapeutic effects of these cells in animal models of glaucoma and other retinal injury models demonstrate their ability to provide neuroprotection for RGCs.
Although numerous recent studies have highlighted the possibility of applying stem cell-based therapy for retinal degenerative diseases, multiple fundamental problems must be resolved before it can be used clinically Few of these methods can be directly transferred to clinical application, considering the obvious side-effects on other retinal resident cells. Therefore, there has been a pressing need to find a suitable cell type and establish some clinically feasible methods if any cell-based therapy is to be considered for inner retinal diseases in the future. The compositions and methods described herein provide a solution to the drawbacks and inadequacies associated with prior stem cell-based approaches for treatment of retinal disease.
As described herein, neural progenitor cells derived from human PFV membranes were successfully cultured and characterized. Although the population of those cells in PFV membrane was relatively small, they stably maintain the same characteristics as progenitor cells and normally proliferate in cultural conditions (
Considering the derivation and location of PFV membranes, studies were carried out to determine whether these membranes contained retinal progenitor cells. Repeated screening for different antigenic markers indicated that undifferentiated NPPFV cells exhibit characteristics of neural progenitors instead of other retinal cell types (Table 2). However, these NPPFV cells could be differentiated in vivo into a retinal ganglion cell-like morphology and express neuronal markers. High expression of β-III-tubulin was observed in undifferentiated NPPFV cells as well. β-III-tubulin is usually considered to be one of the earliest neuron-associated cytoskeletal markers, and plays a significant role in neuritogenesis and cell motility during retinal development. Some studies have revealed that expression of β-III-tubulin could be found in immature neurons of the fetal retina and different neuronal progenitor cells. Some pre-migratory neuroblasts in the postnatal human brain also express β-III-tubulin. Therefore, high expression of β-III-tubulin in undifferentiated NPPFV cells indicates that these progenitors exhibit a migratory phenotype, which facilitates their migration and integration after transplantation.
hRPCs have been isolated from human fetal tissue and well characterized in vitro. The mRNA expression of various transcriptional factors and retinal specific proteins has been detected by PCR in early passages of cultured hRPCs (1). Real-time qRT-PCR results revealed that, compared to hRPCs, NPPFV cells expressed lower mRNA levels of retinal progenitor cell markers and photoreceptor cell markers, but higher mRNA levels of synapse-related protein and THY1 (a mature RGCs marker) (
The neurotransmitter profile of NPPFV cells was examined using Ca2+ imaging analysis, which is widely used to evaluate neural precursors. Unlike neurons from the central nervous system, differentiated NPPFV cells exhibited limited responses to Dopa, Ach, GABA and Gly, but robust responses to Glu (
Cell migration into the uninjured inner retina was observed as early as 1-week and became more significant at 3 weeks post transplantation. This observation indicated that these cells adopted an energetic migratory phenotype after transplantation. As supported by the literature, the number of cells that generally penetrate the retina is usually modest. Millions of cells may be transplanted. However in the examples described here, transplants have been limited to 50,000 cells, which is relatively a small number. Yet the yield of cellular penetration is similar to those studies using many more cells. Some engrafted cells exhibited long and slender projections across the inner plexiform layer. This is the first observation of migration and integration of human cells into uninjured inner retina of adult mice. Of interest, transplanted NPPFV cells seemed to be restricted from migrating into the ONL. Few engrafted cells were found in the outer retina, although sufficient migration was observed in the inner retina. Experiments on subretinal transplantation of NPPFV cells revealed that transplanted cells pooled around the injection site and were restricted from migrating through the ONL. Given that no physical barrier has been reported on the inner side of ONL, it may be possible that the microenvironment in the ONL is inhibitory for NPPFV cell migration. Studies have shown that when progenitor cells are transplanted intravitreally into the adult rodent eye, they do not generally penetrate the retinal barriers and reside on the retinal surface. In order for these cells to penetrate the retina, the retinal barriers need to be broken down. This is achieved by either creating a break in the internal limiting membrane or disturbing Müller/astroglia with glutamate agonists such as α-aminoadipate. The present methods have a distinct advantage over other approached, because NPPFV cells can penetrate the intact inner retina.
Although a plenty of engrafted NPPFV cells were seen in the host retina, the recipient retinal tissue appears morphologically unremarkable with clear structural layers. It is possible that the immunosuppressant effect of rapamycin may have protected the retina, at least partly, from serious xenograft rejection, as severe inflammation usually leads to damage of host tissue after transplantation. The inhibitory effect of rapamycin on mammalian targets of rapamycin pathway may play a role in protecting retina from injuries.
The host retinal environment plays a vital role in neural differentiation of transplanted cells, and additional modulation of retinal environment has been proven to improve migration, integration and differentiation of engrafted cells. Retinoic acid (RA) is an established signaling molecule that is involved in neuronal patterning, neuronal differentiation and the maintenance of the differentiated state of adult neurons and neural stem cells. Embryonic stem cells, hematopoietic stem cells and neural stem cells can be diverted down the neural differentiation pathway using combinations of RA and growth factors, or neurotrophins, which have also been implicated in vivo for their ability to enhance survival and replace lost neurons in the adult brain.
Studies were carried out to test the possibility of inducing differentiation of transplanted NPPFV cells using by RA in vivo. Transplanted NPPFV cells were found to be integrated into the host GCL and exhibited a retinal ganglion cell-like morphology after RA treatment. Expression of synaptophysin was observed, albeit in low frequency, between the connections of differentiated NPPFV cells and host retinal neurons. Similar connections were observed between the engrafted cells and host cells. Although the estimation of the number of differentiated NPPFV cells was very low by visualization of EGFP expression, it is possible that they were underestimated due to the variation of EGFP expression.
However, successful application of RA has proven that it is possible to modify the in vivo environment to enhance the integration and differentiation of transplanted cells in the inner retina. Although NPPFV cells have the potential to differentiate into RGC-like cells, this does not mean that native (in situ) NPPFV cells nested within the retrolental membrane are able to directly migrate from the retrolental membrane into the INL toward areas of retinal pathology without therapeutic intervention. There are many organic and microenvironmental factors that can affect the fate shift and migration of stem cells. Regardless, RA regulates NPPFV cell differentiation in vivo increase the yields and efficiency of inner retina migration and differentiation of NPPFV cells.
Transfection of IGF-1 and IGFBPL1 in Neuronal Progenitor Cells from Human Persistent Fetal Vascular for Neuroprotection
As described above, cells isolated from human persistent fetal vasculature membranes are replete with retinal progenitor cells (hNPPFVs), which differentiate into retinal neurons. In DBA/2J pigmentary glaucoma mice, transplanted hNPPFVs attach and integrated into the inner retinal layer and the optic nerve head (
hNPPFVs were transfected with different plasmids at the concentration of 20 ng/ml. Expression of IGF-1-Tdtomato (
The data from these studies indicated that 1) IGF-1 and IGFBPL1 can be successfully transfected into hNPPFVs; 2) transfected hNPPFVs significantly express and secrete both IGF-1 and IGFBPL; and 3) IGF-1 significantly promotes RGC survival and axonal outgrowth. Thus, increasing IGF-1 and/or IGFBPL1 levels is useful for neuroprotection and promotion of survival and growth of retinal neurons.
As was described above, neuronal progenitor cells from human persistent fetal vasculature incorporate into the retinal ganglion cell (RGC) layer after transplantation. To study whether hNPPFV cells could function as serogate vehicles for local delivery of neuroprotective factors, the effects of IGF-1 were evaluated. RGCs co-cutured with IGF-1-transfected hNPPFV cells displayed significantly enhanced survival, neurite extension and branching, while selective inhibitors of IGF-1 signaling blocked these responses. The findings indicate that transfected hNPPFV cells abundantly deliver IGF-1 and significantly invigorate neuronal survival. The results also indicate that local cell-based delivery of selected neurotrophic factors to protect and rehabilitate host RGCs under disease conditions.
Cells have generally been used as a carrier to load a specific gene to scale up or scale down the expression in a signaling pathway or gene therapy. However, cells used as vehicles in previous studies, unlike hNPPFV cells, have not demonstrated integration and differentiation in host retina after intravitreal transplantation. Based on the advantages of the differentiative potential of hNPPFV cells in the retina and the neurosupportive properties of IGF-1, studies were carried out to test whether hNPPFV cells could be used as candidate vehicles loading igf-1 for continued local delivery of igf-1 in the host retina, whether hNPPFV cells could be stably transfected to express sustained levels of biologically active IGF-1, and whether increase production and secretion of IGF-1 could confer global neuroprotection on RGCs. In this example, igf-1 was cloned into a plasmid carrying a fluorescence reporter gene (tdTomato) to generate fluorescent fusion proteins. The coding sequences of igf-1-tdTomato or tdTomato alone were inserted into a pJ603-neo plasmid backbone. pJ603-neoigf-1-tdTomato generates a fusion protein with tdTomato tagged to the C-terminus of IGF-1. A pJ603-neotdTomato vector generating tdTomato protein was used as a control vector. Transfected cells were studied under co-culture conditions with B6 mouse RGCs and evaluated for their effects on neuronal morphology, apoptosis and neurite growth of RGCs. In addition, we also utilized two IGF-1 antagonists (H-1356 and NBI-31772) with altered affinities for IGF-1 and IGF-1 binding protein, and an antibody to IGF-1R in order to address the neuroprotective mechanisms of IGF-1 signaling pathway. Fragments of IGF-1 that are neuroprotective are identified using the assays described below.
The following materials and methods were used to generate the data described in this example.
Transfection of hNPPFV Cells
hNPPFV cells were thawed from a cell bank. They were previously isolated from human persistent fetal vasculature and were cultured according to established protocols. The coding sequences of igf-1-tdTomato or tdTomato were inserted into a pJ603-neo plasmid backbone (DNA2.0, Menlo Park, Calif.). pJ603-neoigf-1-tdTomato generates a fusion protein with tdTomato tagged to the C-terminus of IGF-1, and pJ603-neotdTomato generates a tdTomato protein alone (used as control vector). Optionally, Gaussia luciferase signal peptide connected at the N-terminus was used to improve IGF-1 expression and secretion. The plasmids were transfected into DH5α Competent E. Coli cells, expanded, and purified using the EndoFree Plasmid Maxi Kit (Qiagen, USA). hNPPFV cells were seeded onto 6-well plates at 1×105 cells/well. The next day, the culture medium in each well was replaced with 1 ml the transfection complex (60 μl Lipofectamine 2000 (Invitrogen); 240 μl plasmid (about 650 ng/ml); and serum-free X-vivo medium). The transfection medium was replaced with regular growth medium after a 5 hr incubation at 37° C. (95% O2, 5% CO2).
hNPPFV cells were transfected with pJ603-neogigf-1-tdTomato or pJ603-neotdTomato in 96-well plates using the same procedure as described above. Two days post-transfection, cells were fixed in 4% paraformaldehyde (20 min), washed in 1×PBS and blocked with the blocking buffer (Li-Cor, Odyssey, Lincoln, Nebr.) at room temperature (30 min). Cells were incubated in primary antibody solution at 4° C. overnight and then rinsed in PBST three times (10 min of each) before being incubated with a secondary antibody solution at room temperature (30 min). After rinsing in PBST twice (10 min in total), cells were visualized under an inverted fluorescence microscope (Olympus 1X51). Primary antibodies used in this study were goat anti-mouse IGF-1 (1:400, B&D systems, MN) and rabbit anti-RFP (1:300, Rockland, Pa.). Secondary antibodies were goat anti-rabbit Cy3 (Chemicon, 1:500) and donkey anti-goat FITC (Chemicon, 1:500).
Transfected hNPPFV cells previously seeded onto 6-well plates were lysed at 48 hr after the transfection and total RNA was extracted using RNeasy Plus Mini Kit (Qiagen, Valencia, Calif.). cDNA was synthesized using the SuperScript® III First-Strand Synthesis System (Life Technologies, USA). 0.5 μl cDNA, 1 μl pre-designed primers of IGF-1 (forward 5′-agatgcactgcagtttgtgtgtgg-3′ SEQ ID NO: 21, reverse 5′-tctacaattccagtctgtggcgct-3′ SEQ ID NO: 22) or GAPDH (forward 5′-ggcctccaaggagtaagacc-3′SEQ ID NO: 23, reverse 5′-aggggtctacatggcaactg-3′ SEQ ID NO: 24), 8.5 μl RNase/DNase-free water and 10 μl KAPA SYBR® FAST reaction buffer (Kapa Biosystems, USA) were loaded into 96-well white PCR plates. qRT-PCR was performed using the Roche LightCycler 480 (Roche LC480, Roche Applied Science) using the following program: 5 min at 99° C., followed by 40 cycles of 15 s at 94° C., 15 s at 59° C., and 15 s at 72° C. Relative amount of igf-1 mRNA expression was normalized to gapdh mRNA (evaluated as ct values) using the well-established delta-delta method. All assays were performed in triplicate. A non-template control was included in the experiment to estimate DNA contamination of isolated RNA and reagents.
Two days post-transfection, hNPPFV cells were lysed to extract total protein using 1×RIPA buffer (Cell Signaling) containing 1 mM phenylmethylsulfonyl fluoride, 1× Protease inhibitor cocktail and 1×EDTA (Thermo Scientific, Rockkford, Ill.). Protein lysates were loaded on 4-20% precise pre-casted PAGE gels (Thermo Scientific, Rockkford, Ill.) and subjected to electrophoresis. Gels were semidry transferred to nitrocellulose membranes (Bio-Rad, Hercules, Calif.) for immuno-blot analysis. Membranes were blocked with blocking buffer for 1 hr at room temperature, incubated with primary antibodies including goat anti-mouse IGF-1 (B&D systems, MN, 1:400) and rabbit anti-GAPDH (1:400, Rochland, Pa.) diluted with the blocking buffer and PBST (volume rate 1:1) overnight (4° C.). Membranes were washed twice in PBST (10 min of each), incubated with secondary antibodies (1:3,000, anti-goat IRDye 800CW and anti-rabbit IRDye 680LT, Li-Cor, Odyssey, Lincoln, Nebr.) for 1 hr, and washed twice in PBST (10 min of each). Fluorescent protein bands were visualized on the Odyssey Infrared Imaging System (Odyssey, Lincoln, Nebr.). After imaging, membranes were briefly striped in stripping buffer (Thermo Scientific), and rinsed in PBST before incubating with rabbit anti-RFP (1:300, Rockland, Pa.) and rabbit anti-GAPDH (1:400) diluted with the blocking buffer and PBST (volume rate 1:1) at 4° C. overnight, followed by secondary antibody incubation. Protein bands were imaged the next day as described above.
Detection of IGF-1 Secretion from Transfected hNPPFV Cells
Secretion of IGF-1 (as component of the IGF-1-tdTomato fusion protein) from hNPPFVigf-1-tdTomato or hNPPFVtdTomato cells was detected using ELISA assay. A 96-well Elispot plate was coated by sodium carbonate buffer (50 μl/well) overnight at 4° C. Conditioned culture medium from hNPPFVigf-1-tdTomato or hNPPFVtdTomato cells (collected on day 1, 3, 5 and 7) and recombinant mouse IGF-1 protein (10-250 ng/ml) were used to coat the Elispot plate overnight (4° C.). After being briefly rinsed, the wells were filled with the blocking buffer (200 μl/well, 10% FBS in 1×PBS) at room temperature for 2 hr. The wells were washed twice with 1×PBS and incubated with goat anti-mouse IGF-1 (1:400) at 4° C. overnight. Wells were washed with 1×PBST three times (5 min each time) and then incubated with chick anti-goat HRP-conjugated secondary antibody (Sigma, 1:5000). Wells were washed twice with 1×PBST and once with 1×PBS. TMB (3,3′,5′5-tetramethylbenzidine) was added to the wells and incubated in dark area for 15-20 min. The reaction was stopped with H2SO4 (2 M) and the plate was quickly read with a Flox4 microarray reader system at OD 450 nm.
P0 B6 mice were euthanized with CO2 and the retinas were dissected out from the eyecups in cold Hank's buffer (Life Technologies) containing 1× Penicillin-Streptomycin-Glutamine (Life Technologies). Retinas were digested in 20 U/ml papain solution containing 100 U/ml DNaseI at 37° C. for 5-15 min; the reaction was stopped with 5 mg/ml ovomucoid protease inhibitor containing 5 mg/ml albumin. Retinas were gently triturated to obtain a single cell suspension, and cells were washed once and re-suspended in 800 μl washing buffer (0.5% BSA, 2 mM EDTA in 1×PBS). RGCs were isolated using Thy1.2 (CD90.2) microbeads and MACS® magnetic separation system (Miltenyi Biotech) following the manufacturer's instructions. RGCs were centrifuged and re-suspended in RGC culture medium (Neurobasal-A medium supplemented with 25 μM L-glutamic acid, 1 mM L-glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 1×B-27, 5 μg/ml insulin, 50 ng/ml BDNF, 50 ng/ml CNTF and 1 μM forskolin) (Life Technologies).
Co-Culture of RGCs with Transfected hNPPFV Cells
hNPPFV cells transfected with pJ603-neoigf-1-tdTomato or pJ603-neotdTomato plasmids were seeded onto cell culture inserts (0.4 μm pore size, BD Falcon) and incubated. On day 3, RGCs were seeded onto 12-well plates pre-coated with Poly-D-Lysine (Millipore, 0.1 mg/ml) and merosin (Millipore, 5 μg/ml), and 200 μl of RGC culture medium was added into each well; culture medium in the inserts containing transfected hNPPFV cells was replaced with 200 μl RGC culture medium before being transferred to the wells. The plates were maintained in an incubator (37° C., 95% O2 and 5% CO2) for three days before being subjected to survival and neurite outgrowth assays. In some experiments, the following reagents were added into the culture medium immediately after RGCs were seeded: IGF-1 receptor antagonist (H-1356, Bachem, 40 μg/ml) which acts as a competitive inhibitor of the IGF-1 receptor, IGFBP (IGF-binding protein) inhibitor NBI-31772 (Millipore, 10 μM), which disrupts the binding of IGF-1 with all six IGFBPs, and a blocking antibody to IGF-1 receptor (IGF-1R, 1:250, R&D Systems).
Co-culture inserts and culture media were removed after 3 days; RGCs were washed with 1×PBS and stained with CalceinAM and EthD-1 (LIVE/DEAD® Viability/Cytotoxicity kit, Life Technologies) for 40 min at room temperature. Images of 4-6 view fields were randomly selected throughout each well under an Olympus inverted fluorescence microscope. Live and dead cells were counted using ImageJ 1.46 (National Institutes of Health, Bethesda, Md.) and survival rates were calculated as live cells/live+dead cells×100%. RGCs in some other wells were fixed with 4% paraformaldehyde for 15 min and then incubated with rabbit anti-mouse β-III Tubulin (Millipore, 1:800) overnight (4° C.) and incubated with goat anti-rabbit Cy3 (1:800) for 1 hr. Images were acquired with an Olympus inverted fluorescence microscope. Neurite lengths were measured using ImageJ.
Statistical analysis was performed using the Sigma Plot. Results are expressed as mean±SD (standard deviation). Differences between groups were compared using Independent Sample t-test. Two-tailed P values <0.05 were defined as significant.
hNPPFVigf-1-tdTomato cells secrete high levels of IGF-1
To investigate the neuroprotective effects of IGF-1 on RGCs and to evaluate whether hNPPFV cells can be used as effective vehicles for IGF-1 delivery, igf-1-tdTomato was introduced into hNPPFV cells by transfecting the cells with plasmid carrying the igf-1-tdTomato fusion gene. In the control group, cells were transfected with a plasmid containing tdTomato. Two days after transfection, over 80% of the cells expressed red fluorescence consistent with tdTomato expression. Immunostaining of the transfected cells with antibodies against tdTomato or IGF-1 confirmed the expression of tdTomato and IGF-1-tdTomato proteins (
To quantify the mRNA levels of igf-1, total RNA was extracted two days post-transfection from hNPPFVigf-1-tdTomato and hNPPFVtdTomato cells and used for quantitative RT-PCR (qRT-PCR). High level of mouse igf-1 mRNA was detected in hNPPFVigf-1-tdTomato cells. In contrast, no mouse igf-1 mRNA was detected in untransfected or hNPPFVTfftdTomato cells (
To identify the expression of IGF-1-tdTomato protein in hNPPFVigf-1-tdTomato cells, whole cell lysates were prepared two days after transfection and used for Western blot analysis. High levels of IGF-1-tdTomato were detected in hNPPFVigf-1-tdTomato cells using antibody against IGF-1, at a molecular weight of 60 kD, agreeing with its predicted molecular weight (about 52 kD for tdTomato portion, and 7.6 kD for IGF-1 portion). TdTomato was detected using antibody against tdTomato in both hNPPFVigf-1-tdTomato and hNPPFVtdTomato cells (
The levels of IGF-1-tdTomato secreted from the hNPPFVigf-1-tdTomato cells were assessed by ELISA using the conditioned culture media collected from the hNPPFVigf-1-tdTomato cells at 1, 3, 5 and 7 days post-transfection. Mouse recombinant IGF-1 protein with concentrations ranging between 10 ng/ml to 250 ng/ml was used to generate the standard curve (as shown in
To evaluate the effects of IGF-1 on RGC survival and neurite outgrowth, primary RGCs were co-cultured from postnatal day 0 (P0) mouse retina with hNPPFVigf-1-tdTomato cells in vitro. We compared survival rates and neurite extension of RGCs co-cultured with hNPPFVigf-1-tdTomato cells with RGCs co-cultured with hNPPFVtdTomato or untransfected cells. The survival rate of RGCs co-cultured with hNPPFVigf-1-tdTomato cells (22±13%) was significantly higher than those co-cultured with hNPPFVtdTomato or untransfected cells (P<0.05;
The average neurite length of RGCs co-cultured with hNPPFVigf-1-tdTomato cells was also significantly longer than those co-cultured with hNPPFVtdTomato or untransfected cells. RGCs co-cultured with hNPPFVigf-1-tdTomato cells produced dramatically long neurites with average lengths of 93±45 μm (P<0.05,
Effects of Inhibitors of IGF-1 Signaling Pathway on IGF-1-tdTomato-Mediated RGC Survival and Neurite Outgrowth
To confirm that the enhancing effects on RGC survival and neurite outgrowth in the hNPPFVigf-1-tdTomato co-cultured group was attributed to activation of the IGF-1 signaling pathway mediated through the IGF-1R, we applied antagonists of the IGF-1 signaling pathway to the culture medium in the co-culture system. H-1356 is an IGF-1 analog, which competitively binds with IGF-1R and blocks IGF-1 signaling. NBI-31772 disrupts the binding of IGF-1 with all six IGF-1 binding proteins. Applying both of these inhibitors completely eliminated the effects of IGF-1-tdTomato on RGC survival and neurite outgrowth (
Previous studies have confirmed the essential roles of IGF-1 on neuron survival and development in the CNS. In the retina, the extended neural structure of the CNS, IGF-1 is a normal constituent playing its role in development. Invtravitreal injections of IGF-1 inhibited secondary cell death of axotomized RGCs in rats. Some in vitro and in vivo studies have showed that IGF-1 is developmentally-regulated and contributes to the visual cortex development. However, these full biological spectrums of IGF1-induced effects on RGCs have remained largely unknown. Moreover, a single intravitreal injection of IGF-1 is short-lasting, and repetitive injections of IGF-1 are needed a considered to be poorly acceptable to patients in clinic. In order to further elucidate the effects of IGF-1 on RGCs as well as to develop a less-invasive, long-lasting and more effective delivery approach, cellular system for delivering biologically active IGF-1 was developed using hNPPFV retinal progenitor cells. The biological effects of IGF-1 on mouse primary RGCs were evaluated in co-culture system, which allowed investigation of the function of IGF-1 on RGC survival and neurite outgrowth, as well as evaluation of the efficacy of using the hNPPFV cells as delivery vehicles for production of IGF-1 to RGCs in vitro. These results indicated that cell-based strategies using hNPPFVs (or NPPFVs from other species) are useful for local delivery of neurotrophic factors into the retina to prevent RGC death and promote optic nerve survival after injury or in disease conditions.
In this example, the igf-1-tdTomato plasmid was successfully transfected into the hNPPFV cells at over 80% transfection rate. qRT-PCR and Western blots confirmed the expression of the transgenes in the hNPPFV cells. ELISA experiments indicated that IGF-1-tdTomato was continuously secreted into the culture medium at high efficiency. In vivo studies of retina have shown that igf-1 mRNA localizes to the ganglion cell layer but no specific localization has been seen in eye sections. In the retina of mammals, IGF-1 plays an essential role during prematurity. Hypodevelopment of vascular growth in retina after premature birth, which is partially due to the insufficiency of IGF-1 expression, causes serve retinopathy leading to visual disorder. A recent study of teleost retina indicates that IGF-1 showed its significant function on regulating rod progenitor proliferation. The data described herein demonstrated that IGF-1 dramatically increased the survival rates and neurite outgrowth of RGCs, and that hNPPFV cells can be used as an efficient delivery vehicle to continuously provide IGF-1.
IGF-1 is primarily synthesized in the liver and plays an essential role in growth and development, and continues to have anabolic effects through adulthood. IGF-1-mediated neuroprotection may involve multiple pathways, including PI-3 kinase and MAPK pathways. The specific receptor of IGF-1 mediated its primary action, which is composed of an extracellular ligand-binding domain that controls the activity of its intracellular tyrosine kinase domain. Mature mouse retinas exhibit decreased the expression levels of p85α regulatory subunit, which was in concurrence with diminished Akt phosphorylation of PI-3 kinase pathway. Furthermore, they provided evidence of retinal and RGC basal lipid kinase activity related with upstream signaling components, such as p85 regulatory and p110 catalytic pathways. The data described herein showed that the survival rate of cultured RGC was remarkably increased in the IGF-1 enriched microenvironment But the survival rate significantly decreased when inhibitors of IGF-1R signaling pathways were applied, which was coincident with previous studies that blocking of IGF-1R signaling pathways induced interruption of survival and proliferation in mitosis-competent cells and growth of tissues. Therefore, binding of newly-secreted IGF-1 to its cognate receptor leads to the cascades of cell signaling resulting to increased survival of RGCs. As described in Example 1, the secreted IGF-1 also affected the morphology of RGCs; specifically, RGCs grew more and longer neurites than those in controls. IGF-1 was also observed to enhance arborization of the neurites in RGCs.
Visual field changes in glaucoma are believed to be caused by the loss of RGCs, although the exact cause of RGC degeneration is still unknown. The aim of neuroprotection for glaucoma therapy is to use agents that prevent or delay retinal ganglion cell death, as well as rescue and enhance regeneration of already compromised RGCs. hNPPFV cells serve as local synthesis pods for local delivery of IGF-1 as they spontaneously integrate into the host RGC and nerve fiber layers after intravitreal injection.
The neuronal progenitor cells and cell lines described herein spontaneously hone in on the RGC and nerve fiber layer and are therefore useful as vehicles for local delivery of a desired neurotrophic factor. The findings indicate that the hNPPFVigf-1-tdTomato cells continuously secret IGF-1-tdTomato, and that the hNPPFV cells efficiently incorporated into the RGC layer after intravitreal transplantation and differentiate into RGC-like cells and survived in the retina over very long period. hNPPFV cells are therefore useful for efficient delivery and supply of neuroprotective factors to prevent RGC death and promote regeneration in vivo in the future.
hNPPFV cells expressing various reporter sequences such as that described above are useful in high-throughput screening for drug discovery and related applications. hPPFVs are neurons and are thus used as substitutes for other neuronal cells in high-throughput assays. Since hNPPFV cells have RGC-like characteristics, they can be utilized in both undifferentiated and differentiated cells.
hNPPFV cells have many advantages compared to other cells used for such assays. For example, primary RGCs are difficult to culture and poorly survive culture conditions necessary for high-throughput screening. Unlike primary RGC cultures, hNPPFV cells have a prolonged survival in tissue culture and can easily accept ectopic reporter gene sequences, which function as reporters for specific cellular activity. hPPFVs may be used as substitute cells for primary RGCs. Therefore, large numbers of molecules may be assayed for neurotrophic activity in hPPFV cells using “live-dead” cells reporter assay. The “hits” can then be further tested on primary RGCs.
Another advantage is the ability to test medium spiny neurons (MSN) pathway function and screen for modulators thereof. An example is determining the intracellular signaling processing of brain neurons such as MSN, which are very difficult to culture and study in vitro. Compounds that influence certain cellular and signaling pathways in MSNs may be subjected to high-throughput study using reporter genes or fluorescent fusion constructs (similar to td-Tomato/IGF-1 described above) that are transfected into hPPFVs. These constructs encoding a detectable marker are used as reporters of cellular pathways of interest. Using the same paradigm, “hits” are then further confirmed on MSN cells.
While the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
The patent and scientific literature referred to herein establishes the knowledge that is available to those with skill in the art. All United States patents and published or unpublished United States patent applications cited herein are incorporated by reference. All published foreign patents and patent applications cited herein are hereby incorporated by reference. Genbank and NCBI submissions indicated by accession number cited herein are hereby incorporated by reference. All other published references, documents, manuscripts and scientific literature cited herein are hereby incorporated by reference.
While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.
This application claims priority to U.S. provisional application Ser. No. 61/645,318, filed May 10, 2012, the contents of which are incorporated herein by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/040556 | 5/10/2013 | WO | 00 |
| Number | Date | Country | |
|---|---|---|---|
| 61645318 | May 2012 | US |
