Invented by Stuart Chambers, Lorenz Studer, Memorial Sloan Kettering Cancer Center

The market for methods of neural conversion of human embryonic derived stem cells is a rapidly growing field in the biotechnology industry. Stem cells have the unique ability to differentiate into various cell types, making them a promising tool for regenerative medicine and drug discovery. Human embryonic stem cells (hESCs) are derived from the inner cell mass of blastocysts and have the potential to develop into any cell type in the body. Neural conversion refers to the process of directing these hESCs to differentiate into neural cells, such as neurons and glial cells, which are essential for the functioning of the nervous system. The demand for methods of neural conversion of hESCs is driven by several factors. Firstly, neurological disorders and injuries, such as Parkinson’s disease, Alzheimer’s disease, and spinal cord injuries, affect millions of people worldwide. These conditions often result in the loss or dysfunction of neural cells, leading to debilitating symptoms. Neural conversion methods offer the potential to replace or repair damaged neural cells, providing hope for effective treatments. Secondly, the development of new drugs for neurological disorders relies heavily on in vitro models that accurately mimic the human nervous system. Neural cells derived from hESCs can serve as a valuable tool for drug screening and toxicity testing, reducing the need for animal testing and accelerating the drug discovery process. The market for methods of neural conversion is diverse, with various techniques and technologies being developed and commercialized. One common approach is the use of small molecules and growth factors to induce neural differentiation. These compounds can activate specific signaling pathways involved in neural development, guiding the hESCs towards a neural fate. Another method involves the use of three-dimensional (3D) culture systems, such as organoids or neurospheres, which mimic the complex architecture and cellular interactions of the human brain. These 3D models provide a more accurate representation of the human nervous system and can be used for disease modeling and drug testing. Furthermore, advancements in gene editing technologies, such as CRISPR-Cas9, have opened up new possibilities for neural conversion. Researchers can now precisely manipulate the genetic makeup of hESCs to enhance their neural differentiation potential or introduce disease-specific mutations for studying neurological disorders. The market for methods of neural conversion is expected to grow significantly in the coming years. According to a report by Grand View Research, the global stem cell market is projected to reach $15.63 billion by 2025, with a compound annual growth rate of 9.2%. The increasing prevalence of neurological disorders, coupled with the growing demand for personalized medicine, is driving the adoption of stem cell-based therapies and neural conversion methods. However, there are several challenges that need to be addressed for the widespread adoption of these methods. One major hurdle is the ethical concerns surrounding the use of hESCs, as their derivation involves the destruction of human embryos. This has led to restrictions and regulations in many countries, limiting the availability and funding for hESC research. Additionally, the scalability and cost-effectiveness of neural conversion methods need to be improved to meet the demands of large-scale applications. The development of standardized protocols and automation technologies can help streamline the process and reduce the variability between different laboratories. In conclusion, the market for methods of neural conversion of human embryonic derived stem cells holds great potential for revolutionizing the treatment of neurological disorders and advancing drug discovery. With ongoing advancements in technology and increasing investment in stem cell research, we can expect to see significant progress in this field in the coming years.

The Memorial Sloan Kettering Cancer Center invention works as follows

The present invention is a general advancement in the field of cell biology, specifically directed differentiation of pluripotent and multipotent cells. This includes human embryonic stem (hESC), somatic (adult) stem cells and induced (hiPSC). Specifically, there are methods for obtaining neural tissues, floor plate cells and placode, including the induction of neural plates development in hESCs to obtain midbrain dopamine neurons (DA), motor neurons and sensory neurons. The neural plate tissue obtained by the methods of this invention can be used in co-cultures to induce differentiation pathways. patterning.

Background for Methods of neural conversion of human embryonic derived stem cells

The differentiation capability of embryonic stem cells and somatic cells has opened up possibilities for cell-replacement therapies for genetic, malignant and degenerative diseases. Cell-based therapies are a promising way to prevent, reduce or eliminate symptoms of neurodegenerative diseases, conditions and disorders. Huntington’s Disease, Alzheimer’s and Parkinson’s are some of these disorders. These cells can also be used to screen for small molecules that are useful for treating disease or determining the fate of neural tissues. These cells were also studied to identify key genes, mRNA transcriptions and proteins that are relevant to normal or pathological cell lineages.

The development of the neural system is governed in space and time by a complex series of signals that determine the identity of the neural precursors.” Although significant progress has been made in animal models of neural development, the human brain is still poorly understood.

Previous studies reported directed differentiation of mouse (Wichterle et al., 2002; Barbed. et al., 2003; Watanabe et al., 2005) and human (Perrier et al., 2004; Li et al., 2008; Eiraku et al., 2008) ESCs into specific neuron types in response to patterning factors defining anterior/posterior (A/P) and dorso-/ventral (D/V) CNS identity. These studies show that signaling systems are conserved across the CNS. SHH is the main ventralizing factor in mammals. It acts in a dose-dependent way to specify various ventral cell kinds, including those that express floor plate (FP), in primary neural explants. (Briscoe & Ericson, 1999). The application of SHH on hESC-derived neurons was shown to induce different ventral neuron types. However, the derivation itself of floor plate tissue (FP) was not reported. As FP is a key signaling center for inducing differentiation pathways, and subsequent cell lineage commitments, the ability of human ES cells to produce FP would be a significant step forward in the study of early human neurodevelopment. Due to the lack of tissue accessibility, we know very little about FP in humans.

In animals, FP is a significant site of SHH and several human development disorders are related (Mullor et. al.,2002) including certain types of holoprosencephaly, microphthalmia and skeletal disorders such as various cleft-plate syndromes and tumor conditions like Gorlin’s Syndrome; a rare genetic condition caused by a mutant in the SHH receptor, Patched 1. It is unknown whether midline SHH signals would cause these diseases in people.

The need to produce human floor plates cells from human embryonic cells is therefore critical. These cells will be used in medical research to determine the causes and treatments for developmental diseases and also for comparative studies on human neural patterning, axonal pathway finding and developmental disorders.

The present invention is a general advancement in the field of stem cell biology, more specifically directed differentiation of pluripotent and multipotent cells. This includes human embryonic stem (hESC), human somatic stem (hiPSC), and induced pluripotent (hiPSC). Specifically, there are methods for obtaining neural tissues, floor plate cells and placode, including the induction of neural plates development in hESCs to obtain midbrain motorneurons and sensory neurons. The neural plate tissue obtained by the methods of this invention can be used in co-cultures to induce differentiation pathways. patterning.

The present invention relates to methods for obtaining neural progenitor cell populations from human embryonic stem (hESCs), and in particular, neural plate tissue. The present inventions are able to induce the development of neural plate floors in hESCs, which is necessary for obtaining dopamine nerve cells (DA). The neural plate floor tissues obtained by the methods of the inventions can be used in co-cultures to induce differentiation pathways. patterning.

The present invention is a general advancement in the field of stem cell biology, specifically directed differentiation of pluripotent and multipotent cells including human embryonic (hESC), human somatic stem (hiPSC), or induced human pluripotent (hiPSC).

The present inventions provide a way to produce a human neuronal cell (neural cells, neuronal types, mature neurons, and cells of a lineage of neural cells) by: (i) obtaining a stem cell (hESCs or hiPSCs or cancer stem cells or human or mammalian multipotent or pluripotent cells); (ii). culturing a human stem cell in conditions that block SMAD signals. In a preferred embodiment of the invention, conditions for culture are included in a feederless system. In a preferred embodiment the stem cells are grown in monolayers. In a preferred embodiment, media containing compounds Noggin or Dorsomorphin as well as SB431542 were used.

In one embodiment, the inventions provide a set comprising a small mothers against decapentaplegic protein signaling inhibitor and a second inhibitor. In one embodiment, the first inhibitor is chosen from a group including a disulfide linked homodimer Noggin (SEQID NO:1), Dorsomorphin LDN-193189 or combining them. In one embodiment, the Noggin can be selected from mouse (human), rat (rat), and xenopus. In one embodiment said Noggin (SEQID NO:1) In another embodiment said second inhibitor inhibits the anaplastic lymphomakinase pathway. In one embodiment, the second inhibitor inhibits a pathway chosen from Lefty, Activin and TGF?. In one embodiment, the second inhibitor inhibits activins as well as nodal signals. In one embodiment, said second inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542) and derivatives thereof. In one embodiment of the kit, a human cell is also included. In one embodiment the kit also includes instructions.

In one embodiment, the inventions provide a compound that includes a stem-cell, a first inhibitor of Small Mothers Against Decapentaplegic protein signaling (SMAD), and a secondary inhibitor of Small Mothers Against Decapentaplegic protein signaling (SMAD). In one embodiment, the first inhibitor is chosen from a group including a disulfide linked homodimer Noggin or Dorsomorphin and LDN-193189. In one embodiment, the is Noggin can be selected from mouse (human), rat (rat), and xenopus. In one embodiment Noggin (SEQ NO:1) is selected from mouse, human, rat, and xenopus. By blocking the phosphorylation ALK4, ALK5 or ALK7 receptors, said second inhibitor inhibits Lefty/Activin/TGF? In one embodiment, the second inhibitor inhibits activin/nodal signals. In one embodiment, said second inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542) and derivatives thereof. In one embodiment, the stem cell selected is from a group including human embryonic stem (hESC), induced human pluripotent (hiPSC) stem cells and somatic stem (SB431542).

In one embodiment, the inventions provide a technique for inducing stem cell differentiation, which comprises: a) providing a cell-culture comprising human cells; ii), a first inhibitor of Small Mothers Against Decapentaplegic protein signaling; iii), a second inhibitor, and b), contacting said cells with said test compound and said first inhibitor, under conditions that induce differentiation of a stem cell to a non default differentiated cell. In one embodiment, the first inhibitor is a disulfide linked homodimer Noggin or Dorsomorphin. In one embodiment, the Noggin selected is from mouse, human rat and xenopus. In one embodiment said Noggin (SEQID NO:1) is used. In another embodiment, the second inhibitor is an ALK4 receptor antagonist. In one embodiment, said second inhibitor is 4-[4-(1,3-benzodioxol-5-yl)-5-(2-pyridinyl)-1H-imidazol-2-yl]benzamide (SB431542) and derivatives thereof. In one embodiment, the non-default differentiated cells is a neural precursor cell. In one embodiment, the non-default differentiated cells are a part a cultured cell population. In one embodiment, the non-default differentiated cells are at least 10% to 100% of the population of cultured cell. In one embodiment, the non-default differentiation cell of a population cultured cells expresses the paired box gene 6. In one embodiment, the paired box 6 protein is expressed by at least 10% in said population of cells. In one embodiment, the stem cell is chosen from the group of human embryonic cells (hESC), somatic cells and induced pluripotent cells (hiPSC). In one embodiment, the non-default differentiated cells is a neuronal cell. In one embodiment, the neural cell is chosen from a group including dopamine-positive neurons and floor platecells.

In one embodiment, the inventions provide compositions comprising isolated neural cells from human embryonic tissue. In one embodiment the isolated human embryonic neurons were derived from embryonic human cells. In one embodiment, the human embryonic neurons are cultured in vitro. In one embodiment the human embryonic cells are attached. In one embodiment of said composition, said co-culture also comprises a second type of cell.

In one embodiment, the inventions provide for a method of screening biological agents. This includes a) providing i) human embryonic stem cell (hESC) culture, and ii), a test substance, and b), contacting said stems with said test compounds under conditions to induce neural floor plate cells. In one embodiment, the test compound is sonic hedgehog or a fragment thereof. In one embodiment the human embryonic stem cell is rosette-stage neurons.

In one embodiment the inventions provide a method for providing differentiated cells, comprising, a) providing i) a cell culture of human embryonic stem cells (hESCs), and ii) a compound for inducing differentiation, and b) contacting said stem cells with said test compound under conditions for inducing neural floor plate cells. In one embodiment, said compound is sonic hedgehog protein or fragment thereof. In one embodiment, said inducing consists of increasing a characteristic selected from the group consisting of flat cellular morphology, expressing sonic hedgehog, expressing forkhead box protein A2 (FOXA2), expressing Netrin-1, and expressing F-Spondin compared to said characteristic expressed in said human embryonic stems cells cultured without said test compound. In one embodiment, said inducing consists of decreasing a characteristic selected from the group consisting of rosette structures, BF1 expression, paired box homeotic gene-6 (PAX6) expression, NK6 homeobox 1 (NKX6.1), homeobox protein SIX6 expression compared to said characteristic in said human embryonic stems cells cultured without said test compound. In one embodiment, the method further provides and comprises a Noggin protein and an agent for blocking phosphorylation of a receptor selected from the group consisting of activin receptor-like kinase 4 (ALK4), activin receptor-like kinase 5 (ALK5) and activin receptor-like kinase 7 (ALK7) receptors and contacting said human stem cells with said noggin and said agent to human stem cells before adding said compound. In one embodiment, the method further provides an antibody, wherein said antibody is dickkopf homolog 1 (DKK-1) antibody, and contacting said stem cells with said antibody for reducing DKK-1 protein function. In one embodiment, the method further provides and comprises a caudalizing factor selected from the group consisting of wingless-type MMTV integration site family, member 1 (Wnt-1), and Retinoic Acid (RA). In one embodiment, the method further provides and comprises a neuron inducing compound and step c) adding said neuron inducing compound for inducing progenitor neurons. In one embodiment, said dopamine neurons express a marker selected from the group consisting of corin, serine peptidase (CORIN) and nephroblastoma overexpressed gene (NOV). In one embodiment, said progenitor neurons are dopamine neurons express a marker selected from the group consisting of LIM homeobox transcription factor 1, ? (LMX1B) and neurogenin 2 (NGN2). In one embodiment, the method further provides and comprises a stem cell, and step d) co-culturing said human neural floor plate cells with said stem cells for producing neurite outgrowth from said stem cells.

In one embodiment, the inventions provide neural floor plate cells produced by the described methods. In one embodiment, the inventions provide an elastomeric cell made by the described methods. In one embodiment, the inventions provide lens cells produced by the described methods.

The invention consists of methods for assessing neural identity in the derived neurons. The method can be morphological, functional, or based on the expression of proteins that are associated with certain lines. “A preferred method is to use dopaminergic activities or motor neuron functional assays.

The present method is a powerful way to deliver neural cells or agents to the brain for treatment, diagnosis, and prevention of diseases, disorders or patients with nerve damage from stroke. These cells were committed to a neural fate.

In one embodiment, “the present invention contemplates compositions comprising human embryonic cells isolated from the floor plate.” In one embodiment, isolated human embryonic cell floor plates were derived from embryonic human cells. In one embodiment, human embryonic cells were cultured in vitro. In one embodiment the human embryonic cells of the floor plate are attached cells. In one embodiment the composition is also a coculture that includes a second type of cell.

In one embodiment, this invention is a method of screening biological agents. It comprises a) providing a cell-culture comprising human embryonic cells (hESCs) and ii), a test substance, and b), contacting said stems with said test compounds under conditions to induce neural floor plate cells. In one embodiment, sonic hedgehog fragment or a test compound is used. “In one embodiment, human embryonic stem cell rosette-stage neurons are used.

In one embodiment, this invention envisages a method of providing differentiated cell, which comprises a) providing i) human embryonic stem (hESC) cells and ii), a compound to induce differentiation; and b), contacting said stems with said test compound in conditions that will cause neural floor plate cells. In one embodiment, it is sonic-hedgehog protein or a fragment thereof. In one embodiment, inducing is performed by increasing a characteristic from the group consisting flat cellular morphology (sonic hedgehog), forkhead protein A2(FOXA2) expression, Netrin-1 expression, and F-Spondin expression compared to the characteristic expressed in the human embryonic stem cells cultured without the test compound. In one embodiment, inducing is a process of decreasing a characteristic from the group consisting BF1 expressions, paired box gene-6 (PAX6), NK6 Homeobox 1(NKX6.1), and homeobox proteins SIX6 compared to the characteristic expressed in said human stem cells cultured without the test compound. In one embodiment, a Noggin protein is provided along with an agent that blocks phosphorylation at a receptor chosen from the group consisting activin-like receptor-like-kinase 4, activin-like receptor-like-kinase 5, and activin-like receptor-like-kinase 7, and the method includes contacting human stem cells using said noggin, and the agent, before adding the compound. In one embodiment, a method is provided that further comprises an antibody, namely dickkopf homolog 1 antibody (DKK-1), and contacting stem cells with the antibody to reduce DKK-1 function. In one embodiment, a caudalizing agent is provided in addition to the method. The group of factors includes wingless-type MMTV family member 1 (Wnt-1), Retinoic acid (RA), and wingless type MMTV integration sites. In one embodiment, step c) adds said neuron-inducing compound to induce progenitor neurones. In one embodiment, dopamine neuronal cells express a marker chosen from the group consisting corin, serine-peptidase gene (CORIN), and nephroblastoma expressed gene (NOV). In one embodiment, progenitor cells are dopamine neuronal express a mark selected from the group consisting LIM homeobox transcript factor 1. (LMX1B), and neurogenin 2, (NGN2). In one embodiment of the method, it also includes providing stem cells and step d), co-culturing human neural floorplate cells with said stem cell to produce neurite outgrowth.

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