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  • C12N5/0602—Vertebrate cells
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  • C12N5/0619—Neurons
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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• • A—HUMAN NECESSITIES
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  • C12N2501/30—Hormones
  • C12N2501/38—Hormones with nuclear receptors
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  • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
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  • C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes

Definitions

• MNPs Motor Neuron Progenitors
• the present invention relates to producing motor neuron progenitors (MNPs) from human induced pluripotent stem cell (iPSC) lines and human embryonic stem cell (hESC) lines. More particularly, this invention provides a method of producing greater than 75-90% purity and functional MNPs from various hESC lines and iPSC lines.
• the present invention also relates to cryopreserving MNPs. More particularly, this invention provides a method of cryopreserving MNPs that allows more than 90% recovery of highly viable and functional cells post-thawing.
• Neurons may be classified based on their structure and function. Structural classification is based on the number of processes extending from the neuronal cell body. In contrast, functional classification is based on the direction in which the neuron transmits nerve impulses.
• Motor neurons are efferent neurons that convey nerve impulses from the brain and spinal cord (that is, away from the central nervous system) to effectors (which may be either muscles or glands).
• the satellite- shaped cell body of the MN is connected to a single, long axon (which forms a neuromuscular junction with effectors) and several shorter dendrites projecting out of the cell body.
• MN function and associated diseases such as amyotrophic lateral sclerosis (also known as ALS or Lou Gehrig's disease) and spinal muscular atrophy.
• ALS amyotrophic lateral sclerosis
• iPSCs iPSCs
• an object of the present invention is to provide a method of producing MNPs from human pluripotent cells (including hESCs and iPSCs) that is simple, efficient, scalable, and reproducible.
• a further object of this invention is to provide a method of cryopreserving MNPs that allows the frozen cells to be thawed with high viability and functionality.
• a further object of this invention is to provide methods applicable to pluripotent stem cells in general, and induced pluripotent stem cells in particular.
• a further object of this invention is to provide universal methods applicable regardless of cell source.
• a further object of this invention is to provide for higher robustness and viability of MNPs. [0012] A further object of this invention is to provide for greater than 90% viability of recovered MNPs.
• a further object of this invention is to reduce the clumping of the cells.
• a further object of this invention is to provide a method of cryopreserving MNPs that allows the frozen cells to be thawed with high recovery.
• a further object of this invention is provide a method including the use of mouse embryonic fibroblast (MEF) feeder cells to culture the iPSCs and hESCs
• a method for producing MNPs in vitro by harvesting hESCs or iPSC, which have been grown on mouse embryonic fibroblasts for at least 6-7 days, and these undifferentiated hESCs or iPSCs are neuralized by plating and culturing in ultra-low- adherence flasks containing a serum-free motor neuron induction medium for about 5 days.
• the induction medium is a classical medium containing growth factor, non-essential amino acids, L-glutamine, insulin, transferrin, selenium and B27 and is supplemented with bFGF and retinoic acid to generate spheres.
• the cultured spheres are ventralized by using an induction medium supplemented with low concentration of bFGF for about 10 days to promote formation of neurospheres.
• the suspension cultured neurospheres are mechanically dissociated into smaller spheres and expanded on an adherent surface for about 5 days as neural rosettes or early stage MNP. Thereafter, the adherent early stage MNPs are dissociated using trypsin solution and are transferred to gelatin-coated flasks containing induction medium to further enrich MNP cells.
• the non-adherent cells are collected from the gelatin-coated flasks, re-plated as adherent cells in matrix-coated flasks, such as Matrigel®, containing induction medium, and repeatedly cultured and re-fed with induction medium for about 5-6 days. Then, the adherent late stage MNPs are harvested from the matrix-coated flasks using trypsin solution. The resulting cell suspension is transferred to gelatin-coated flasks to further enrich MNP cells and remove contaminant cells. Non-adherent MNPs from the gelatin-coated flasks are collected, and large cell clumps are sedimented in a conical tube. MNPs are collected from the supernatant in the conical tube following the sedimentation of the large cell clumps can be stored as cryopreserved MNPs.
• the concentration of bFGF is used at 10 ng/ml in the induction medium for the first day of plating of undifferentiated hESC or iPSC through day 7 of culture, and bFGF concentration is decreased to 5 ng/ml on day 8 of culture until harvesting of the MNPs.
• the concentration of retinoic acid is used at 10 ⁇ from day 1 through day 7 culture.
• the harvested MNPs-containing supernatant is centrifuged and the resulting cell pellet of MNPs is re- suspended in a cold protein-free and serum-free freezing medium pre-formulated with DMSO in a cryovial.
• a cold protein-free and serum-free freezing medium pre-formulated with DMSO in a cryovial.
• An example of such freezing media can include CRYOSTORE® CS10 solution, by BioLife of Seattle, WA.
• 10% DMSO can also be used and optimized to freeze cells.
• the cryovial is transferred to a controlled rate freezer and subjected to programmed freezing process. Thereafter, the cryovial is transferred from the controlled rate freezer to a liquid nitrogen Dewar for long term storage.
• the present invention overcomes a major disadvantage of current methods of producing MNPs for therapeutic applications by providing a simple, highly efficient, scalable, and reproducible method of differentiating MNPs from various lines of hESCs and iPSCs.
• One invention disclosed herein is the use of mouse embryonic fibroblast (MEF) feeder cells to culture the iPSCs and hESCs.
• MEF mouse embryonic fibroblast
• hESCs were originally derived and cultured on MEF layers which permit continuous growth of hESCs in an undifferentiated stage (Amit et al 2003; Biology of Reprod 68:2150-2156). In vitro, the hESCs tended to differentiate when cultured in the absence of MEF feeder layers (Thomson et al 1998 Science 282 (5391) 1145-7). Feeder cells have also been derived from several human cell types such as human foreskin fibroblasts or adult Fallopian type epithelial cells (Amit et al 2003 Biology of Reproduct 22(5) 1231-8; Richard et al 2002 Nat Biotech 20(9) 933-6; Richards et al 2003. Stem Cells 21(5)546-56; Hovatta et al 2003.
• Mouse feeder cells express more Activin A than human feeder cells, but they do not express bFGF like human feeder cells (Eiselleova et al 2008. J of Devel Biol 52(4) 353- 6315). When compared to human feeder cells, MEFs seem to support better the undifferentiated growth of some hESC lines, whereas more spontaneous differentiation and a lower proportion of SSEA3 positive cells can be observed with human feeder cells (Eiselleova et al 2008. J of Devel Biol 52(4) 353-6315). Cultured feeder cells secrets numerous of uncharacterized growth factors, cytokines, extracellular matrix (ECM) components such as proteoglycans, fibronectin, various types of collagen, nidogen, and laminin.
• ECM extracellular matrix
• ECM proteins, growth factors and cytokines secreted by feeder cells provide hESC/iPSCs a scaffold for hESC/iPSCs to anchor and provide the signals to proliferate and maintain their pluripotency.
• the present inventors have unexpectedly found that using feeder cells maintains the pluripotent state of the iPSCs and hESCs such that these cells are primed in better conditions for subsequent neuralization and ventralization steps of motor neuron differentiation process.
• This important change in methodology over the currently available art improves neuralization potency and results in a functional and more homogenous population of MNPs.
• the method of the present invention can be used for a variety of iPSC and hESC lines with consistent yield of high purity MNPs. Accordingly, the method of the present invention has significant improvements over the current technology that requires culturing of hESCs under feeder cell-free conditions such as matrix gel (U.S. Patent Number 8,137,971).
• the present invention also introduces a highly efficient freezing method for MNPs that includes the use of chemically-defined cryoprotectants and a controlled rate freezer to improve cell recovery after thawing from long term storage.
• the latter improvement over the prior art allows the long term storage of MNPs, which provides greater experimental flexibility in downstream applications. Cryopreservation during the differentiation process introduces efficiencies for the commercial manufacture of MNPs.
• FIGURE 1 illustrates an MNP manufacturing process.
• FIGURES 2A-E illustrates: (A) cultured iPSCs on MEFs feeder; (B) neurospheres in suspension culture condition after caudalization on day 8; (C) neurospheres in suspension culture after ventralization on day 18; (D) Plated neurospheres on adherent substrate showing migration of the early MN progenitors; and (E) expansion of the early MN progenitors after first purification.
• FIGURES 3A-C provide images of characterization of MNPs before cryopreservation on day 28: (A) MNP specific marker Islet 1 ; (B) MNP specific marker HB9; and (C) neurofilament protein( Tuj 1).
• FIGURES 4A-C illustrate images of MN progenitors after cryopreservation and re- plated on PDL/laminin coated surface on day 3 after thawing:
• A Thawed MN progenitors after cryopreservation with branched morphology;
• B neurofilaments (Tuj l, green) and GFAP ( red) and DAPI nuclear staining (blue);
• C HB9 (red) transcription factor makers for MN progenitor and neurofilament (Tuj l, green).
• FIGURE 5 illustrates maturation of MNP in the absence and presence of B27 in laminin coating step.
• MNPs Provided herein are methods for the production of MNPs from various lines of hESCs and iPSCs.
• Equipment list laminar flow biosafety cabinet class II, centrifuge, water bath, incubator, refrigerator, first freezer, second freezer, pipet aid or pipet ball, multi-channel pipettors, multichannel aspirator, microscope, assorted pipettors and pipet tips, hemocytometer, NucleoCounter ® (Lonza), Cell scrappers (Corning 3010),T-25 flasks (Corning 430639),T-75 flasks (Corning 430641), T-75 ultra-low adherence flasks (Corning 3814), 15 ml centrifuge tubes (BD Falcon 352097),50 ml centrifuge tubes (BD Falcon 352098), aspirating pipets (BD Falcon 357558), assorted serological pipets (BD Falcon 356543, 357551, 357525, 357550), 50 ml Steriflip ® tubes (Millipore SCGP00525), 50 ml reagent reservoirs, precision balance, assorted Nalgene ® bottles (Fi
• the water bath is about 37°C.
• the incubator is capable of maintaining 37°C + 2°C with a 5% + 2% C0 2 and humidified atmosphere.
• the refrigerator is capable of maintaining about 2 to 8°C.
• the first freezer is capable of maintaining about -20 to -30°C.
• the second freezer is capable of maintaining about -78 to - 82°C.
• the hESC medium is Knockout DMEM supplemented with 20% KOSR, glutamax, non-essential amino acids and bFGF and beta mercaptoethanol, MNP Induction medium such as a 50:50 mixture of classical DMEM high glucose and DMEM/F12 supplemented with insulin, transferrin, selenium, glutamine, magnesium chloride and B27 (NSF1). Once mixed, the medium was used for no more than 14 days.
• MNP Induction medium such as a 50:50 mixture of classical DMEM high glucose and DMEM/F12 supplemented with insulin, transferrin, selenium, glutamine, magnesium chloride and B27 (NSF1).
• the MNP basal medium is a classical DMEM high glucose supplemented with B27 (NSF1), non-essential amino acids, insulin, transferrin, selenium, hepes, magnesium chloride, zinc sulfate, and copper sulfate with or without L-glutamine. Once mixed, the medium was used for no more than 14 days.
• NSF1 B27
• non-essential amino acids insulin, transferrin, selenium, hepes
• magnesium chloride zinc sulfate
• copper sulfate copper sulfate
• the MNP Plating medium is MNP basal medium supplemented with B27 (NSF1) and L-glutamine.
• the MNP maintenance medium is MNP basal medium supplemented with B27 (NSF1).
• low osmolity medium is KnockOut DMEM/F12 (Gibco 12660-012).
• the an optimized, cGMP produced, protein-free and serum- free freezing medium pre-formulated with DMSO is CryoStor® CS10 (BioLife® Solution 210102).
• the laminin working solution is stock laminin solution (500 ⁇ g/ml) diluted by mixing 300 ⁇ of stock laminin solution in 10 ml of MNP basal medium (15 ⁇ ⁇ ).
• ten sterile cryovials were labeled and stored at -20°C, 30 minutes prior to use.
• Five mg of Poly-D-Lysine powder from Sigma was rehydrated in 10 ml of water for cell culture for at least 30 minutes in the laminar flow hood.
• the rehydrated poly-D- lysine stock solution (500 ⁇ g/ml) was aliquoted at 1 ml/vial and the aliquots were stored at -20°C until required for coating.
• a 1 ml aliquot of the stock Poly-D-Lysine solution was thawed and diluted by mixing it in 9 ml of PBS.
• the working solution of poly-D-lysine 50 ⁇ g/ml was used for coating.
• coUagenase IV working solution is 0.1 ml/cm2 of 1 mg/mL coUagenase solution (7.5 ml per T-75 flask and 2.5 ml per T-25 flask).
• An appropriate amount of milligrams of coUagenase powder was weighed out using the precision balance by multiplying by 2 the calculated volume of coUagenase solution.
• the weighed out coUagenase was transferred into a 50 ml tube and the calculated volume of low osmolarity DMEM/F12 medium, e.g. KnockoutTM DMEM/F12 from Life Technologies, was added.
• the solution was mixed thoroughly by swirling until all the coUagenase was completely dissolved, and was filtered through a 0.22 ⁇ filter before use.
• the solution was stored at 4° C and used within one week.
• aliquots were prepared in the laminar flow hood as quickly as possible with the hood lights turned off.
• the 100 mg vial of retinoic acid was disinfected and placed it in the laminar flow hood.
• the top of the glass vial was broken off carefully and discarded into a sharps bin.
• DMSO dimethylsulfoxide
• the rinses were transferred to the 50 ml tube containing the retinoic acid mixture. Another 12.6 ml of DMSO were added to the tube and mixed well by pipetting up and down several times. This made a stock solution of 20 mM retinoic acid. Using a 200 ⁇ pipette, 100 ⁇ aliquots of stock solution were made in 500 ⁇ amber tubes and the tubes were placed immediately in a -80°C freezer.
• hESCs or iPSCs were co-cultured with MEF feeder cells with hESC growth medium (knockout DMEM/F12, 20% KSR, Glutamax, NEAA, BME and bFGF) on the T75 flask(FIGURE 2A).
• hESCs were initiated when the cell density reached around 80% confluence.
• Spent medium from the T-75 flask was replaced with 30 ml of a 1: 1 mixture of hESC medium and MNP induction medium supplemented with 10 ng/ml bFGF.
• the hESCs/iPSCs colonies were dissociated using a collagenase solution (1 mg/ml) and the dissociated cells were suspended with MNP induction medium supplemented with 10 ng/ml bFGF and 10 ⁇ of retinoic acid. The cell suspension then was transferred into an ultra-low- adherence T-75 flask.
• retinoic acid was removed from medium and bFGF concentration was reduced to 5ng/mL. Numerous neurospheres were formed while some have the tendency to attach to other cells and form larger cellular spheres (FIGURE 2C). Medium was replaced every other day until day 20 by using the same procedure but at a shorter sediment time to remove non- neurospheres.
• the spheres along with the spent medium were transferred into a 50 ml conical tube.
• the spheres were allowed to sediment for about 30 seconds and the spent medium was aspirated.
• the spheres were resuspended in 5-7 ml fresh medium and they were sedimented for about 15-30 seconds while spent medium was aspirated. This washing step was repeated twice.
• the spheres were pipetted gently with a 10 ml serological pipet to break up the aggregates and were transferred to Matrigel®-coated T-75 flasks and distributed evenly prior to placing in the incubator.
• the cultures were dissociated with TrypLE solution. Once the cells were dissociated, to each T-75 flask, 15 ml of MNP induction medium supplemented with bFGF was added and the cell aggregates were triturated gently to break up the remaining spheres. The cell suspension was centrifuged for 3 minutes at 200 xg. The cells were resuspended with 10 ml of fresh MNP induction medium supplemented with bFGF and the suspension was transferred into the gelatin-coated T-75 flask. The T-75 flask was incubated for 15 minutes at 37° C undisturbed.
• MNPs (FIGURE 2E) were harvested by using the same methods as described in previous section of Day 25 MNP using trypLE and purified by using gelatin coated flasks to remove non MNP cells. MNPs were collected as non-adherent cells from gelatin coated flasks. Cell counts and viability were determined by using NucleoCounter.
• FIGURE 1 provides an overview of the MNP differentiation process.
• a chemically defined formulation is introduced: 50:50 mixture of classical DMEM high glucose and DMEM/F12 supplemented with insulin, transferrin, selenium, glutamine, magnesium chloride and B27 (NSF1).
• NSF1 non-adherent aggregates condition
• RA retinoic acid
• MNPs are characterized with neuronal marker and MNPs specific markers before cryopreservation. The results showed that on day 28, majority cells expressed HB9, Isletl and Tuj l (FIGURE 3). PSCs marker such as Oct4 and glial marker GFAP were not detectable. Expression of mesodermal marker SMA was very minimal.
• T-75 flasks were coated with 7 ml per flask of 0.1% gelatin solution and incubated for 30 minutes in the incubator. Approximately 150 ml of MNP induction medium were pre-warmed. The spent medium from the T-75 flasks was aspirated and the flasks were washed once with 15 ml of PBS each. Five ml of TrypLE solution were added and incubated 3-10 minutes. The flask was examined every 3 minutes until most cells were dissociated. Ten ml of MNP induction medium were added to each flask and the suspension was transferred into 50 ml conical tubes.
• Each T-75 flask was rinsed with an additional 10 ml of medium and the solution was transferred to the 50 ml tubes.
• the tubes were centrifuged at 200 x g for 3 minutes at room temperature. The supernatant was aspirated and the cell pellet was resuspended in 20 ml of medium and the suspension was transferred into the gelatin-coated T-75 flasks (10 ml/flask).
• the conical tubes were rinsed with 4 ml of MNP induction medium and the solution was transferred to the gelatin-coated T-75 flask. The T-75 flask was incubated for 15 minutes at 37°C undisturbed.
• the supernatant (>95% of the solution) was transferred into a fresh tube and the cells were counted using a NucleoCounter®.
• the tubes were centrifuged at 200 x g for 3-5 minutes at room temperature.
• the supernatant was aspirated and 10 ml of cold CryoStor® solution were slowly added to the cell pellet and the cell pellet was gently resuspended. About 100-200 ⁇ of suspension were taken for counting again using NucleoCounter®.
• the remaining cell solution was put on ice while waiting for cell counts.
• the viable cell concentration was adjusted to 6 million/ml by adding more CryoStor® solution, and 1 ml suspension was aliquoted into each cryovial.
• a programmable controlled rate freezer was used to freeze MNPs using the following parameters. After the freezing cycle was completed, cryo vials were transferred to liquid nitrogen Dewar immediately after the freezing program was completed [0070] Results
• cryoprotectant was not utilized and cooling rate might not be consistent due to a number of reasons such as location within the - 80C mechanical freezer.
• cryoprotectants were examined together with controlled rate freezer to ensure the cooling rate was consistent.
• cryoprotectants such as trehalose, mannitol, and hetastarch were used in combination with DMSO and compared with CryoStor CS10 (Table 1).
• Each 96 well plate required 10 ml of poly-D-lysine working solution (50 ⁇ g/ml). The required amount of poly-D-lysine working solution was transferred into a sterile reagent reservoir. Using a multi-channel pipettor, each well of a 96 well plate was coated with 100 ⁇ of poly-D-lysine working solution and the plates were placed in the incubator overnight. After incubation, the poly-D-lysine solution was aspirated using a multi-channel aspirator. The wells were rinsed twice with 100 ⁇ per well of PBS. The plates were dried in the laminar flow hood with the lids off for at least 1 hour and were ready for laminin coating.
• the required amount of laminin working solution (15 ⁇ g/ml) was prepared in MNP basal medium (without B27/NSF1). Using a multi-channel pipettor, 75 ⁇ of laminin working solution were added per well. The plates were incubated at 37 °C for at least one hour but no longer than six hours. The laminin solution was aspirated when the cells were ready to plate. The wells were rinsed with 100 ⁇ of medium per well once prior to plating the cells.
• the supernatant was aspirated, the cells were resuspended in 2 ml of fresh plating medium, and 200 ⁇ of cell suspension were counted in the NucleoCounter®. The number of cells required at 40,000 viable cells per well and the cell suspension required for plating were calculated. The calculated cell suspension to have a final concentration of 40,000 viable cells per 300 ⁇ of plating medium was resuspended.
• the cell suspension was transferred to a sterile reservoir and using a multi-channel pipet 300 ⁇ of the cell suspension were added per well of the MNP.
• the 96 well plates were placed in the incubator. The plates were left undisturbed for at least 24 hours.
• laminin coating step used a medium that containing B27. This step created and mixture of B27 and laminin that compete with each other to bind to poly-D-lysine surface and yield a non- homogenous surface for MNP plating. It is well-known that neurons in general are plated on the laminin and poly-D-lysine surface. Our hypothesis is B27 present in laminin coating step causes the MNP clump.
• a maturation MNP experiment was carried out using either poly-D-lysine coated with laminin alone or laminin+B27 mixture. The cultures were monitored at various time points during the maturation process. The results showed that after 10 days, clumping process started to appear in the culture of Laminin+B27 (FIGURE 5D). The clumping became more apparent in MNP culturing on laminin+B27 after 17-21 days (FIGURE 5F) while there was no clump detected in culture with laminin coating alone (FIGURE 5C and FIGURE 5E). These results were consistent with our hypothesis that the B27 is the cause for the MNP clumping during the maturation process. EXAMPLE 4
• a batch record was initiated at Day 0 for every non-clinical MNP initiation and was maintained by the production department. Quality records were retained for GMP and ISO requirements as specified in internal standard operating procedures for the retention of quality records. Applicable ISO standards were followed and/or referenced.

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