A method using charged surfaces to generate multiple lineages from induced pluripotent stem cells

Abstract

This disclosure provides a method for generating progenitor cells from induced pluripotent stem cells, wherein the progenitor cells include endothelial cells, pericytes, brain microvascular endothelial cells (BMEC), mesenchymal stem cells (MSC), hematopoietic progenitor cells (HPC), microglia, or neural progenitor cells (NPC).

Description

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 861,640, filed June 14, 2019; No. 62 / 865,806, filed June 24, 2019; and No. 63 / 038,564, filed June 12, 2020, the entire contents of which are incorporated herein by reference.

[0002] background

[0003] 1. Field

[0004] This invention generally relates to the fields of molecular biology and medicine. More specifically, it relates to a method for differentiating induced pluripotent stem cells.

[0005] 2. Relevant Technical Specifications

[0006] Human pluripotent stem cells (hPSCs) offer a powerful resource for applications in regenerative medicine and drug development. Over the past decade, various methods have been developed to generate multiple lineages from iPSCs using a variety of growth components, extracellular matrix, and / or feeder layers, providing potentially powerful tools for improving in vitro models and studying the early development of different lineages.

[0007] Effective in vitro differentiation into specific cell types is crucial for their potential applications in disease modeling and drug screening. Key success factors for lineage-specific differentiation include the use of defined conditions, favorable phenotype and functional characterization of terminally differentiated cells, reproducibility of the differentiation process length, and cost. Therefore, there remains a need in the art for effective methods for the specific differentiation of human pluripotent stem cell lineages.

[0008] Overview

[0009] Certain embodiments of this disclosure provide methods and compositions for an in vitro method of differentiating induced pluripotent stem cells (iPSCs), including: (a) culturing iPSCs on a charged surface in the absence of extracellular matrix proteins; and (b) differentiating iPSCs into endothelial cells, mesenchymal stem cells (MSCs), or hematopoietic progenitor cells (HPCs).

[0010] In some respects, the charged surface is positively charged. In some respects, the positively charged surface is an amine surface or a poly-L-lysine surface. In certain respects, the positively charged surface contains nitrogen-containing functional groups. In other respects, the charged surface is negatively charged. In certain respects, the negatively charged surface is a carboxyl surface. In some respects, the negatively charged surface contains oxygen-containing functional groups. In some respects, the charged surface is a polymer surface. For example, the polymer surface is a polystyrene surface. In some respects, the charged surface contains both positively charged and negatively charged groups. In some respects, the positively charged group is a nitrogen-containing group, and the negatively charged group is an oxygen-containing group.

[0011] In some respects, iPSCs are cultured in serum-free, component-specific media. In other respects, differentiation involves culture in the presence of blebbistatin or a ROCK inhibitor such as H1152. In specific respects, the method is free of or substantially free of extracellular matrix proteins such as laminin, fibronectin, hylocinin, and matrigel. TM Tenosynovin, nestin, platelet-reactive protein, elastin, gelatin or collagen.

[0012] In another aspect, the method further includes engineering the iPSCs prior to step (a) to have disrupted expression of TREM2, MeCP2, and / or SCNA. In a particular aspect, the engineering includes introducing an insertion or deletion in exon 2 of TREM2 using a TAL nuclease. In some aspects, disrupted MeCP2 expression is further defined as a truncated mutant of the MeCP2 protein. In some aspects, the disrupted expression is due to a missense point mutation such as A53T.

[0013] In some aspects, the method includes differentiating progenitor cells into endothelial cells. In some aspects, step (a) includes culturing on an amine surface to generate progenitor cells and step (b) includes culturing on a carboxyl surface in the presence of an endothelial differentiation medium to generate endothelial cells. In a particular aspect, the endothelial cells are CD31 positive.

[0014] In a further aspect, the method further includes differentiating endothelial cells into brain microvascular endothelial cells (BMEC).

[0015] In some respects, the method further includes differentiating endothelial cells into lymphatic endothelial cells.

[0016] In some respects, the method involves differentiating progenitor cells into MSCs. In certain respects, differentiation includes culturing progenitor cells on an amine surface in the presence of MSC medium. In some respects, MSCs are positive for CD73, CD44, and CD105. In some respects, at least 90% of the differentiated cells are positive for CD73.

[0017] In another aspect, the method further includes differentiating MSCs into pericytes. In some aspects, MSCs are cultured in the presence of a pericyte medium in the absence of extracellular proteins. In some aspects, the pericytes are positive for NG2, PDGFRβ, and CD146.

[0018] In some aspects, the method includes differentiating progenitor cells into HPCs. In some aspects, the method also includes differentiating HPCs into microglia. In some aspects, differentiation includes culturing HPCs on a neutrally charged or ultra-low adhesion surface in the presence of a microglia differentiation medium. In some aspects, the microglia differentiation medium contains IL34, TGF, and MCSF. In some aspects, differentiation includes culture under normoxic conditions. In some aspects, differentiation lasts for 20–25 days. In certain aspects, the microglia are positive for CD45, CD11b, and CD33. In some aspects, at least 50% (e.g., 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 50–60%, 60–70%, or 80–90%) of the differentiated cells are positive for CD11b. In some respects, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the differentiated cells are positive for CD33.

[0019] In some respects, this method does not include cell purification. In other respects, purification is further defined as performing MACS.

[0020] In certain respects, this method conforms to Good Manufacturing Practices (GMP). In some respects, this method is carried out under hypoxic conditions. In specific respects, iPSC is human.

[0021] In another embodiment, a composition comprising a microglia population is provided, wherein at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 90-93%, 93-96%, or 96-100%) of the microglia population is positive for P2RY12, CX3CR1, TMEM119, IBA-1, and TREM2. In some aspects, the microglia population is generated by a method according to the embodiments of the present invention. In some aspects, the microglia population is generated from a disease-associated iPSC donor having TREM2, APOE, CD33, BIN, ABCA7, SNPS, or a genotype associated with neurodegeneration. In some aspects, the microglia population has disrupted expression of TREM2, MeCP2, and / or SCNA. In some aspects, disrupted TREM2 expression is further defined as homozygous knockout of TREM2 expression. In some respects, disrupted MeCP2 expression is further defined as a truncated mutant of the MeCP2 protein. In other respects, disrupted SCNA expression is caused by missense point mutations such as A53T.

[0022] Another embodiment provides a method for screening test compounds, comprising introducing the test compound into a microglia population according to an embodiment of the invention. In some aspects, the microglia population is further introduced into amyloid-β. In some aspects, the microglia population is further introduced into LPS.

[0023] Another embodiment provides a composition comprising a pericyte cell population generated by the method of this embodiment.

[0024] In yet another embodiment, this document provides a blood-brain barrier model comprising microglia, pericytes, and BMECs generated by embodiments of the present invention.

[0025] Another embodiment provides a method for generating microglia, comprising: (a) differentiating iPSCs into HPCs; (b) sorting HPCs against CD34-positive cells; and (c) culturing HPCs in a microglia differentiation medium to generate a microglia population. In some aspects, HPCs are differentiated according to embodiments of the invention. In some aspects, sorting includes the use of CD34 magnetic beads. In certain aspects, the method does not include sorting HPCs against CD43-positive cells. In certain aspects, the method does not include ECM proteins.

[0026] In some cases, microglia differentiation media contain IL-34, TGFβ1, or M-CSF. In others, the microglia differentiation media contain 200 ng / mL IL-34, 100 ng / mL TGFβ1, and 50 ng / mL M-CSF. In still others, cells are fed with microglia differentiation media every 48 hours.

[0027] In certain respects, at least 90% (e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 90-93%, 93-96%, or 96-100%) of the microglia population are TREM-positive. In some respects, at least 10% (e.g., 15%, 20%, 25%, 30%, 10-15%, 15-20%, or 20-30%) of the HPCs differentiate into microglia.

[0028] In some respects, the cultivation in step (b) is performed in a 96-well format. In some respects, the cultivation in step (b) is performed on a charged surface. In some respects, the charged surface is positively charged. For example, a positively charged surface is an amine surface. In other respects, the charged surface is negatively charged. For example, a negatively charged surface is a carboxyl surface.

[0029] In another aspect, the method further includes maturing microglia in a culture medium containing CD200 and / or fractal chemokines. In some aspects, the method further includes cryopreservation of microglia.

[0030] In some respects, HPCs differentiate from iPSCs engineered to disrupt TREM2 expression. In other respects, the engineering involves the use of TAL nucleases.

[0031] In some respects, cryopreserved microglia retain their phagocytic function against pHrodo bacterial particles. In other respects, cryopreserved microglia are able to mature upon thawing, respond to stimuli, and secrete interleukins, chemokines, and immunomodulatory ligands in the culture supernatant.

[0032] Another embodiment provides an in vitro method for generating neural progenitor cells (NPCs) from iPSCs, comprising (a) pre-conditioning iPSCs in a medium containing an inhibitor of glycogen synthase kinase 3 (GSK3); and (b) differentiating iPSCs into NPCs, wherein the method does not involve inhibiting SMAD signaling.

[0033] In some respects, the iPSCs are maintained under hypoxic conditions prior to step (a). In other respects, the iPSCs are inoculated in the presence of a ROCK inhibitor prior to step (a), and then cultured in the absence of a ROCK inhibitor.

[0034] In some respects, the GSK3 inhibitor is CHIR99021, BIO, or SB-216763. In specific respects, the GSK3 inhibitor is CHIR99021, for example, at concentrations of 1 μM, 2 μM, 3 μM, 4 μM, or 5 μM, particularly 3 μM. In some respects, the pretreatment lasts for 2–4 days, for example, 1, 2, or 3 days.

[0035] In some respects, the iPSCs of steps (a) and / or (b) are cultured on a surface coated with extracellular matrix (ECM) proteins. In some respects, the ECM proteins are MATRIGEL. TM Laminin or hyalin. Specifically, ECM proteins are laminins or hyalin.

[0036] In some respects, steps (a) and (b) are performed under normoxic conditions. In some respects, differentiation involves culturing iPSCs on a surface coated with ECM proteins. In some respects, the ECM proteins are laminin or telonelin. In some respects, differentiation involves culturing iPSCs on ultra-low adhesion plates or spin flasks in the presence of ROCK inhibitors. In certain respects, step (b) is performed for 5 to 10 days, such as 6, 7, 8, 9, or 10 days.

[0037] In another aspect, the method also includes detecting the expression of Tra-162, CD56, CD15, Sox1, neural epithelial stem cell protein, β3 microglobulin, and / or Pax-6 in NPCs. In some aspects, at least 70% (e.g., 80%, 85%, 90%, 95%, 70-80%, 80-90%, or 90-100%) of NPCs are positive for CD56.

[0038] In a further aspect, the method includes further differentiating NPCs into astrocytes or neurons.

[0039] Another embodiment provides a method for screening neurodegenerative diseases, including detecting the level of soluble TREM2 in a microglia conditioned medium. In some aspects, the detection includes performing an ELISA. In some aspects, the microglia are derived from genetically engineered iPSCs or donors expressing disease-related SNPs or mutations. In some aspects, the microglia are generated by methods according to embodiments of the present invention or aspects thereof. In some aspects, an increased level of soluble TREM2 compared to a control is used to detect neurodegenerative diseases such as Alzheimer's disease or multiple sclerosis.

[0040] Another embodiment provides a method for performing high-throughput screening to identify therapeutic agents, comprising contacting microglia generated by a method according to embodiments of the present invention or aspects thereof with a variety of candidate agents and measuring cytokine and / or chemokine levels and / or amyloid β phagocytic function.

[0041] In some respects, the microglia are cryopreserved microglia derived from genetically engineered iPSC lines, microglia derived from donors expressing disease-related SNPs, or microglia derived from donors expressing mutations associated with neurodegeneration. In some respects, the cytokines and / or chemokines are selected from IL6, IL10, IL3, TNFα, IL13, CCL2 / MCP-1, CCL20 / MIP-3α, CCL4 / MIP-1β, CCL5 / RANTES, CX3CL1 / fractal chemokine, CXCL1 / GROα, CXCL10 / IP-10, CXCL2 / GROβ, and IL-8 / CXCL8.

[0042] This document also provides co-cultures of microglia and endothelial cells, pericytes, astrocytes, and / or neural progenitor cells comprising embodiments and aspects thereof. Another embodiment provides the use of this co-culture in simulating human brain development.

[0043] Other objects, features, and advantages of the invention will become apparent from the following detailed description. However, it should be understood that while the detailed description and specific embodiments indicate preferred embodiments of the invention, they are given by way of example only, as various changes and modifications within the spirit and scope of the invention will be apparent to those skilled in the art based on this description.

[0044] Brief description of the attached figures

[0045] The following figures form part of this specification and are included to further illustrate certain aspects of the invention. A better understanding of the invention can be achieved by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein.

[0046] Figure 1 Schematic diagram of the differentiation process of 2D and 3D HPC.

[0047] Figure 2 : A schematic diagram of endothelial cells derived from HPCs on day 6 of the differentiation process originating from 2D or 3D HPCs.

[0048] Figure 3 : Without using CD31 + Characterization of endothelial cells generated by sequential passage purification in the context of MACS.

[0049] Figure 4A -B:( Figure 4A The morphology of cells at the end of replate passage 3. Endothelial cells can be cryopreserved at the end of replate passage 2 or 3. Figure 4B (A culture medium formulation for deriving endothelial cells.)

[0050] Figures 5A-5D :( Figure 5A An overview of the MSC differentiation process. Figure 5B (This refers to) the culture medium formulation used to produce MSCs. Figure 5C This diagram illustrates the three lineages of MSC differentiation: adipocytes, osteocytes, and cartilage cells. Figure 5D Phenotypic characterization of MSC progenitor cells on day 6.

[0051] Figure 6 Pure MSC populations appeared on the amine surface. Cryopreserved HPC on day 6 or live cultures at the end of differentiation on day 6 were placed on amine-charged plates in MSC medium and the presence of 1 μM H1152 (or blebbistatin). At P4, the cultures were transferred to normoxic and normal tissue culture plates. MSC purity specifications were achieved at P5.

[0052] Figure 7Cells stained for the absence of surface MSC markers CD73, CD44, CD105, CD49d, and endothelial markers CD31 and CD144 were used. Cryopreserved day 6 HPC or live cultures at the end of differentiation on day 6 were placed on amine-charged plates in MSC medium and the presence of 1 μM H1152 (or blebbistatin). At P5, the cultures were transferred to normoxic and normal tissue culture plates. MSC purity specifications were achieved at P6.

[0053] Figures 8A-8C :( Figure 8A Tri-lineage potential. The various steps involved in the generation of adipocytes, osteocytes, and chondrocytes from MSCs. Figure 8B Images showing the trilineage potential of MSCs, Alizarin Red staining of osteocytes, Alcian Blue staining of chondrocytes, and Oil Red O staining of adipocytes. Figure 8C MSCs at 1,000 cells / cm 2 Inoculate into MSC differentiation medium and feed every other day for 10–14 days. Stain plates with crystal violet and count total colonies.

[0054] Figures 9A-9H MSCs transform into pericytes. Figure 9A A schematic diagram illustrating the process of converting iCell MSCs into iPSC-derived pericytes. Figure 9B (A culture medium formulation for producing iPSC-derived pericytes.) Figure 9C Comparative flow cytometry analysis of known pericyte markers PDGFRβ, NG2, and CD146 in iCell MSCs, iPSC-derived pericytes, and ScienCell primary human cerebral vascular pericytes (HBVP). iCell MSCs showed no pericyte markers at thawing, but these markers were obtained at the end of P1 in the pericyte culture medium. iPSC-derived pericytes exhibited higher purity of pericyte-specific markers than primary HBVP. Figure 9D Morphological examination of iPSC-derived MSCs (P2), iPSC-derived pericytes (P1), and primary HBVP cells of ScienCell using bright-field microscopy. Figure 9E A table describing the differences between PC1 and PC2 pericyte subtypes based on phenotype and biomarker expression. Figure 9F iPSC-derived pericytes were stained by flow cytometry immediately after thawing and five days later for pericyte subtype-specific markers CD274, VCAM1, desmin, DLK1, and αSMA, as well as universal pericyte markers PDGFRβ, NG2, CD13, and CD146. iPSC-derived pericytes revealed the characteristic features of the contractile pericyte PC2 subtype. Figure 9GImages of iPSC-derived pericytes using the IncuCyte real-time imaging system in a phagocytosis assay. iPSC-derived pericytes show observable phagocytic activity of Staphylococcus aureus bioparticles at levels higher than the control. (A) iPSC-derived pericytes alone (control). (B) iPSC-derived pericytes + NucGreenDead 488 (NG) reagent (control). (C) iPSC-derived pericytes + Staphylococcus aureus pHrodo Red bioparticles (BP). (D) iPSC-derived pericytes + NucGreenDead 488 reagent (NG) + Staphylococcus aureus pHrodo Red bioparticles (BP). All images were taken at a time point of 36 days and 16 hours post-cell seeding. Figure 9H The overall intensity of red objects was analyzed using IncuCyte software to quantify phagocytic activity.

[0055] Figure 10A-10G The production of brain microvascular endothelial cells (BMECs). Figure 10A A schematic diagram illustrating the generation of brain microvascular endothelial cells. Figure 10B Composition of ECRA medium. Figure 10C Flow cytometry analysis of BMECs by co-expression of Glut1 / CD31. Figure 10D Immunohistochemical staining of BMECs expressing P glycoprotein (green) on day 13. Cell nuclei were stained with Hoechst 3342 and images were captured at 200x magnification using ImageXpress (Molecular Devices, LLC). Figure 10E BMEC was functionally characterized by measuring the TEER value several days after panel placement. Figure 10F A schematic overview of an alternative method for generating brain microvascular endothelial cells (BMECs), including pretreatment steps and plate-coating on a charged surface without the use of an ECM. A description of the modified culture medium for inducing BMEC generation on a charged surface. Figure 10G Differentiated brain microvascular endothelial cells were harvested on different charged surfaces on day 7, and purity was quantified by staining for the presence of CD31, p-glycoprotein / Glut-1 expression and the absence of expression of the pluripotency marker (TRA-181).

[0056] Figure 11 : Scale up HPC using a 3D differentiation process, followed by CD34 +Purification is performed using magnetic beads. A schematic diagram illustrates the proportional addition and sorting of HPCs using CD34 magnetic beads. Examples of the efficiency of the sorting process via manual and CliniMACs-mediated separation are provided. The actual purity of HPCs per run (measured by the percentage of CD34-positive cells in the sorted fraction) and the efficiency of the process are summarized.

[0057] Figure 12 : Culture medium formulation for microglia differentiation.

[0058] Figures 13A-13B :( Figure 13A A schematic diagram of HPC-derived microglia sorted from CD34+ cells. HPC cells were plated in MDM microglia differentiation medium. Cultures were fed with MDM or 2X MDM every 48 hours. Cultures were split on day 12 of differentiation, and 2D differentiation continued until day 23. Cells were harvested on day 23 and stained for the presence of markers of microglia purity. The remaining cultures were cryopreserved. The purity of microglia cultures was quantified before and after cryopreservation. Figure 13B The microglia differentiation culture medium and the microglia differentiation culture medium were described.

[0059] Figure 14 Microglial cell purity was assessed in the presence of MDM medium before and after cryopreservation. Microglial cell cultures at day 23 of differentiation were harvested and stained for the presence of microglial-specific markers. The remaining cells were cryopreserved using a rate-controlled cryostat. The cryopreserved cells were thawed and stained for the presence of microglial-specific markers. For both groups, cell surface expression of CD45, CD33, TREM2, and CD11b was assessed by flow cytometry. Figure 14 A) Intracellular expression of CX3CR1PU.1, IBA, P2RY12, TREM2 and TMEM119.

[0060] Figures 15A-C: Microglia recovery after cryopreservation using a manual relative rate-controlled cryostat. HPCs were placed in culture medium to initiate microglia differentiation in the presence of MDM. Cells were cryopreserved on days 20 (Figure 15B), 23 (Figure 15B), and 26 (Figure 15C) using either a manual cryopreservation protocol or a rate-controlled cryostat (CRF). The cryopreserved cells were transferred to liquid nitrogen for one week. The cryopreserved microglia were thawed and placed in microglia maturation medium (MMM). The culture was fed fresh maturation medium every 48 hours. Cells were harvested on days 3, 5, 7, 10, 12, and 14 post-thawing, and viable cell recovery relative to the initial plate number was quantified.

[0061] Figure 16 Efficiency of HPC conversion to microglia. Cryopreserved HPCs differentiated into microglia in the presence of MDM (N=4). The total number of viable input HPCs and output microglia was quantified. Process efficiency was calculated by dividing the purity and absolute number of TREM2-positive cells present on day 23 of microglia differentiation by the absolute number of input viable HPCs.

[0062] Figures 17A-17C Purity analysis of microglia cryopreserved manually or in the presence of a rate-controlled freezer on days 20 (Fig. 17A), 23 (Fig. 17B), and 26 (Fig. 17C), 3 days and 10 days post-thawing. Microglia cryopreserved on days 20, 23, and 26 were thawed and plated in microglia maturation medium for 3, 5, 7, 10, and 12 days. Cells were stained by flow cytometry for the presence of Pu1, IBA, CX3CR, and P2RY12 expression.

[0063] Figures 18A-18B : Manually count live cells of cryopreserved microglial cells on day 0 (Fig. 18A) and day 3 (Fig. 18B) after thawing, or in the presence of a rate-controlled freezer, to set up a phagocytosis assay using Staphylococcus aureus bioparticles.

[0064] Figure 19 Functional characterization of microglia cryopreserved on days 20, 23, and 26 of differentiation using manual or rate-controlled cryopreservation. Cryopreserved microglia were thawed and seeded at 15,000 viable cells / well in 96-well plates with 200 μl of microglia maturation medium per well. Cells were treated with diluted 1 μg / well of conditioned or unconditioned pHrodoRed bioparticles (Thermo Fisher #A10010, 2 mg per vial; stored at -20°C). Plates were placed on IncuCyte, and images of phagocytosis were captured at different time points up to 5 days after thawing. Cells cryopreserved using the rate-controlled cryopreservation method exhibited stronger phagocytosis (due to higher cell viability). Manual cryopreservation showed a decreasing / rightward shift in phagocytosis rate under all conditions (due to decreased cell viability).

[0065] Figure 20Functional characterization of live day 14 microglia and microglia cryopreserved at days 20, 23, and 26 of differentiation using manual or rate-controlled cryopreservation, assessed by real-time imaging on the IncuCyte system. Cryopreserved microglia were thawed and plated in MMM for three days. Viable cell counts at the end of three days were determined as shown in Figure 18B. 15,000 viable cells were plated into 96-well plates in the presence of 200 μl of microglia maturation medium (MMM) per well. Cells were fed 50 μl of fresh MMM every 48 hours. Cells were treated with diluted 1 μg / well of conditioned or unconditioned pHrodo Red bioparticles (Thermo Fisher #A10010, 2 mg per vial; stored at -20°C). Plates were placed on the IncuCyte system, and images of phagocytosis were captured at different time points at 5, 7, and 14 days after thawing. The manual cryopreservation method shows the rate of decrease / right shift in phagocytosis (due to reduced cell viability) under all conditions.

[0066] Figure 21 The phagocytic efficiency ratio is determined by dividing the phagocytic red blood cell count from the thawed sample by the total number of cells.

[0067] Figure 22 Functional characterization of cryopreserved microglia using pHrodo amyloid β. Microglia frozen for 23 days were thawed and seeded at 15,000 viable cells / well in 96-well plates with 200 μl of microglia maturation medium per well. Cells were treated with pHrodo amyloid β. The control group consisted of cells with medium containing no pHrodo amyloid β. Plates were placed on IncuCyte, and images of phagocytosis were captured at different time points up to 24 hours after thawing.

[0068] Figures 23A-23B Miniaturization of HPC differentiation into microglia in the absence of ECM and in a 96-well format suitable for screening applications. Schematic diagram of HPC differentiation into microglia (Fig. 23A) and different charged surfaces used in the experiments. Ultra-low adhesion (ULA), tissue culture (TC), and non-tissue culture (Non-TC) containers (Fig. 23B).

[0069] Figures 24A-24B Analysis of the late-stage purity of microglia on day 23 in the presence of various charged surfaces. Cryopreserved HPCs were analyzed at 20,000-35,000 viable cells / cm³. 2Cells were seeded at a density of 200 μl of microglia differentiation medium per well on 96-well Primaria plates or ultra-low attachment, tissue culture (TC) or non-tissue culture (Non-TC) plates. During the next 23 days of differentiation, cells were fed with 50 μl of MDM medium per well every 48 hours. Cells were harvested on day 23 with cold PBS, and the total viable cell count was quantified using an automated cell counter. Cells were stained for surface expression of CD11b, CD45, CD33, and TREM2, and for intracellular expression of TREM2, IBA, P2RY12, and TMEM119.

[0070] Figures 25A-25B Cytokines and chemokines released from cryopreserved microglia. Microglia cryopreserved on day 23 were thawed into MDM medium and seeded at 50,000 cells / well in Primaria 96-well plates. Cells were seeded for three days prior to stimulation with 100 ng / ml LPS and 50 ng / ml interferon-gamma. Stimulation was performed in triplicate and lasted 24 hours. The supernatant was centrifuged to remove cells and debris and immediately placed at -20°C. The supernatant was analyzed on a multiplex Luminex assay. Multiple WT batches are shown. Figure 25A ) and WT, homozygous and heterozygous TREM2 knockout (KO), MECP2 HM and A53T-SNCA engineered systems ( Figure 25B The average value of ).

[0071] Figure 26 Cryopreserved microglia from multiple batches of homozygous and heterozygous TREM2 knockout (KO), MECP2, and A53T-SNCA engineered lines were stained for surface expression of CD11b, CD45, and TREM2, as well as the presence of intracellular markers PU.1, IBA-1, CX3CR1, P2RY12, and TMEM119. Engineered iPSC lines showed comparable TREM2 expression by flow cytometry.

[0072] Figures 27A-27B :( Figure 27ARelease of soluble TREM2 from cryopreserved microglia derived from wild-type (WT), heterozygous (HT), and homozygous (HO) TREM2 KO engineered iPSCs. Soluble TREM2 (sTREM2) levels were quantified from conditioned media derived from WT and TREM2 heterozygous and homozygous KO mutants using a Simple Step ELISA (AbCam). WT and TREM2 KO microglia were thawed and plated at the same density in maturation medium in 96-well Primaria plates. Used medium was collected on days 3 and 7 post-thaw. Cultures were fed semi-feeded with fresh maturation medium on days 3 and 5 post-thaw. Figure 27B Release of soluble TREM2 from cryopreserved microglia derived from wild-type (WT), MECP2HM, and A53T-SNCA engineered iPSCs. The levels of soluble TREM2 (sTREM2) were quantified from conditioned media derived from WT, MECP2HM, and A53T-SNCA engineered lines using a Simple Step ELISA (AbCam). Microglia were thawed and plated at the same density in mature medium in 96-well Primaria plates. Used medium was collected on days 3 and 7 post-thaw. Cultures were fed semi-feeded with fresh mature medium on days 3 and 5 post-thaw.

[0073] Figure 28 List of culture medium formulations used to study the survival kinetics of WT, HT, and HO TREM2 KO engineered microglia (WT, 1185HT TREM2 KO, and 1187HO TREM2 KO). Microglia were placed at a density of 15,000 live cells per well in 250 μl of microglia basal medium containing 32 different cytokine formulations in 96-well plates. Cell survival kinetics were captured on the IncuCyte system. NucGreen Dead diluted to 2 drops / mL was added to all wells containing cells based on different culture medium compositions to capture the number of dead cells over time. Images were taken every 8 hours, and the experiment lasted for 72 hours without any intermittent feeding.

[0074] Figures 29A-29C Microglial cell survival kinetics of WT (Fig. 29A), 1185HT TREM2 KO (Fig. 29B), and 1187HO TREM2 KO (Fig. 29C).

[0075] Figures 30A-30C Microglial cell survival kinetics using two cytokines: WT (Fig. 30A), 1185HT TREM2 KO (Fig. 30B), and 1187HOTREM2 KO (Fig. 30C).

[0076] Figures 31A-31C Microglial cell survival kinetics using three cytokines: WT (Fig. 31A), 1185HT TREM2 KO (Fig. 31B), and 1187HOTREM2 KO (Fig. 31C).

[0077] Figures 32A-32C Microglial cell survival kinetics using four cytokines: WT (Fig. 32A), 1185HT TREM2 KO (Fig. 32B), and 1187HOTREM2 KO (Fig. 32C).

[0078] Figures 33A-33E WT, 1185HT TREM2 KO, and 1187HO TREM2 KO microglia were placed at a density of 15,000 viable cells in 250 μl of microglia basal medium (Fig. 33A) or MMM (Fig. 33B), or microglia basal medium supplemented with IL-34 (Fig. 32C), or microglia basal medium supplemented with IL-34 (Fig. 33D), or microglia basal medium supplemented with MCSF (Fig. 33D), or basal medium supplemented with IL-34 only (Fig. 33C), or MSCF, or a combination of IL-34 and MCSF (Fig. 33E). Cell viability kinetics were captured on the IncuCyte system. NucGreen Dead diluted to 2 drops / mL was added to all wells containing cells with different culture medium compositions to capture the number of dead cells over time. Images were taken every 8 hours, and the experiment continued for 7 days without any intermittent feeding. The intensity of NucGreen Dead quantified the number of dead cells in the culture.

[0079] Figures 34A-34H Functional characterization of pHrodo Red-labeled bacterial bioparticles and pHrodo Red amyloid β in WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia cryopreserved on day 23. WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia were thawed at a concentration of 15,000 viable cells / cm³. 2Cells were plated at density in 250 μl MMM (Fig. 34A-B) or supplemented with MDM matrix (AKA microglia matrix medium) supplemented with MSCF alone (Fig. 34C-D), IL-34 (Fig. 34E-F), or a combination of IL-34 and MCSF (Fig. 34G-H) in 96-well plates for three days. Cells were treated with diluted 1 μg / well of conditioning or non-conditioning pHrodo bioparticles (Thermo Fisher #A10010, 2 mg per vial; stored at -20°C) (Fig. 34A, C, E, G) or pHrodo amyloid β (Fig. 34B, D, F, H). Plates were placed on IncuCyte and images of phagocytosis were taken at different time points up to 30 hours. WT and engineered microglia showed phagocytic function after thawing. Kinetics and phagocytic efficiency differed among WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia.

[0080] Figure 35 This study aimed to simplify the purity of microglia cultures in maturation media. The purity of wild-type (WT) microglia cryopreserved on day 23 was determined after thawing in maturation media in the presence of MMM or microglia basal medium supplemented with a combination of two key cytokines (IL-34 and MSCF). Purity was quantified by harvesting cells on days 3, 7, and 14 post-thawing, and the purity of CD45, CD33, TREM2, CD11b, CX3CR1, P2RY12, TMEM119, and IBA was determined by harvesting cells at the end of differentiation. Cell surface staining and intracellular marker staining were performed by flow cytometry. Cryopreserved microglia maintained viability and purity in maturation media supplemented with MSCF and IL-34. This simplified medium would be valuable for the co-culture application of cryopreserved microglia with neurons and astrocytes for the development of brain organoid TREMs.

[0081] Figures 36A-36B :( Figure 36A A schematic diagram illustrating a screening experiment using cryopreserved microglia. Figure 36B A table of compounds tested during the screening.

[0082] Figures 37A-37D Using GW501516 ( Figure 37A Leucettine L41 Figure 37B ), leucine ( Figure 37C ) and Azeliragon ( Figure 37D The results of the microglia screening experiment.

[0083] Figures 38A-38D Using J147 ( Figure 38A ), dibutyryl-cAMP ( Figure 38B ), Iradipine ( Figure 38C ) and besalotine ( Figure 38D The results of the microglia screening experiment.

[0084] Figures 39A-39E Using SB-431542 ( Figure 39A SP600125 Figure 39B ), GW2580 ( Figure 39C ), PP2 ( Figure 39D ) and SB239063 ( Figure 39E The results of the microglia screening experiment.

[0085] Figure 40A-40D : Use GW501516 in the presence of LPS stimulation ( Figure 40A Leucettine L41 Figure 40B ), leucine ( Figure 40C ) and Azeliragon ( Figure 40D The results of the microglia screening experiment.

[0086] Figures 41A-41E : Use J147 in the presence of LPS stimulation ( Figure 41A ), dibutyryl-cAMP ( Figure 41B ), Iradipine ( Figure 41C ), Besarotin Figure 41D ) and SB-43152 ( Figure 41E The results of the microglia screening experiment.

[0087] Figures 42A-42D SP600125 can be used in the presence of LPS stimulation. Figure 41A ), GW2580 ( Figure 42B ), PP2 ( Figure 42C ) and SB239063 ( Figure 42D The results of the microglia screening experiment.

[0088] Figure 43 Summary of microglia screening experiment results.

[0089] Figure 44A-44G :( Figure 44A The microglia-ratio with all traces of ATP / BzATP. Figure 44B Microglial cell ratio with ATP / BzATP sample trace. Figure 44C The response of microglia to BzATP. Figure 44D Differential responses to ADP in microglia. Figure 44E The response of 100 μMBz ATP in the presence of the P2X7 antagonist AZ11645373. Figure 44F The response of 100 μMBz ATP in the presence of the P2X7 antagonist A438079. Figure 44G Dose-dependent response to demonstrate in AZD1283 (P2Y) 12 Functional ADP-dependent response in microglia in the presence of an effective receptor antagonist.

[0090] Figures 45A-45B Using Simple Step ELISA (AbCam) from day 3 after thawing ( Figure 45A ) or the 7th day ( Figure 45B The conditioned medium collected was quantitatively derived from the release of soluble TREM2 from cryopreserved microglia of ANH and disease-associated microglia. ANH and DAM-associated microglia were thawed and plated at the same density in mature medium in 96-well Primaria plates. Used medium was collected on days 3 and 7 post-thaw. Cultures were fed semi-feeded with fresh mature medium on days 3 and 5 post-thaw.

[0091] Figures 46A-46J Cytokines and chemokines released from cryopreserved microglia derived from a group of iPSC donors with ANH and disease-associated microglia. Cryopreserved microglia were thawed in MDM medium on day 23 and seeded at 50,000 cells / well in Primaria 96-well plates. Cells were seeded for three days before stimulation with 100 ng / ml LPS or IL-4 + dBu-cAMP. Stimulation was performed in triplicate over 24 hours. The supernatant was centrifuged to remove cells and debris and immediately placed at -20°C. The supernatant was analyzed on a multiplex Luminex assay. Following LPS stimulation, M1 analyte (… Figure 46A M2 analyte ( Figure 46B ), interleukin ( Figure 46C ), chemokines ( Figure 46D ) and other analytes Figure 46E The release of ) . After stimulation with IL-4+dBu-cAMP, the release of analyte M1 ( Figure 46F M2 analyte ( Figure 46G ), interleukin ( Figure 46H ), chemokines ( Figure 46I ) and other analytes Figure 46J The release of ).

[0092] Figure 47A-47JDemonstrating phagocytic function of AHN and disease-associated microglia using pHrodo-labeled Staphylococcus aureus bioparticles and amyloid β: Cryopreserved AHN and disease-associated microglia were seeded at 5,000 cells per well in 384-well poly-D-lysine plates. Staphylococcus aureus bioparticles were added to each well at a concentration of 0.5 μg / mL, and amyloid β was added at a concentration of 1 μM / well. The phagocytic kinetics of Staphylococcus aureus bioparticles and amyloid β were quantified using the total red matter intensity using the IncuCyte live cell analysis system. Figure 47A (ANH, Staphylococcus aureus), ( Figure 47B (ANH, β-amyloid protein), ( Figure 47C (R47H is relative to ANH, Staphylococcus aureus), ( Figure 47D (R47H relative to ANH, amyloid β), ( Figure 47E (CD33 relative to ANH, Staphylococcus aureus), ( Figure 47F (CD33 relative to ANH, amyloid β), ( Figure 47G (ABCA7 relative to ANH, Staphylococcus aureus), ( Figure 47H (ABCA7 relative to ANH, amyloid β), ( Figure 47I (APOE isotype relative to ANH, Staphylococcus aureus), ( Figure 47J (APOE isotype relative to ANH, amyloid β).

[0093] Figure: 48A-48D:( Figure 48A A schematic diagram illustrating a method for generating neural progenitor cells (NPCs) from iPSCs without the use of dual SMAD inhibition. The individual steps involved and the composition of the culture medium used are described. Figure 48B This study summarizes the kinetics of NPC emergence in three iPSC lines. It examines the decrease in pluripotency markers and the appearance of NPC-specific markers at different days during differentiation. Purity was quantified by cell surface staining and intracellular staining by flow cytometry. Figure 48C Staining of astrocytes derived from multiple passages of NPC cells in cultures. Quantification of astrocyte purity was achieved by flow cytometry through staining of the cell surface and intracellular surfaces. Figure 48D NPCs were differentiated into neurons, and the purity of terminal neurons was quantified by intracellular flow cytometry.

[0094] Figure 49 Summary of surfaces compatible with iPSC-derived cell lineages.

[0095] Description of exemplary implementation schemes

[0096] In some embodiments, this disclosure provides methods for generating multiple lineages of cells (e.g., endothelial cells, mesenchymal stem cells (MSCs), and hematopoietic progenitor cells (HPCs)) from induced pluripotent stem cells (iPSCs). Typically, this method involves differentiating iPSCs into various lineages using a charged surface. Specifically, the differentiation method can be performed in the absence of extracellular matrix (ECM) proteins and allows for self-purification through passage, for example, for generating MSCs and endothelial cells.

[0097] In a further embodiment, methods are provided for differentiating endothelial cells into brain microvascular endothelial cells (BMECs) or lymphatic endothelial cells, differentiating MSCs into pericytes, and differentiating HPCs into microglia. This process allows for the efficient generation of endothelial cells and MSCs without the need for purification, such as MACS purification or the use of ECM proteins. The process can be adapted to Good Manufacturing Practice (GMP) compliance.

[0098] Endothelial cells form a network of interconnected cells in the human body, lining blood vessels, lymphatic vessels, and forming capillaries. Endothelial cells regulate the flow of nutrients and produce and respond to various bioactive molecules, offering potential applications in tools for screening compounds and drugs targeting vascular toxicity, vascular permeability, and therapeutic uses (including treatment of tissue ischemia and bioengineered grafts). Numerous protocols exist for deriving endothelial cells. Almost all processes require magnetically activated cell sorting (MAC) separation using CD31 microbeads to generate pure cultures of endothelial cells.

[0099] In some embodiments, this disclosure provides a method for generating a pure endothelial cell population from iPSCs (e.g., additionally reprogrammed iPSCs) via a two-step process. The iPSC cells can be converted into hematopoietic progenitor cells (HPCs) on a positively charged amine surface, then further proliferate and subsequently purify the endothelial cells in the presence of a negatively charged carboxyl surface. The endothelial cells can be derived from hematopoietic endothelial cells without any MAC purification steps. These cells express CD31 / CD144 / CD105 at high purity and are capable of proliferating to maintain purity and cryopreservation at both early and late passages.

[0100] Mesenchymal stem cells (MSCs) isolated from adult tissues are capable of proliferating in vitro and maintaining their pluripotency, making them an attractive cell source for regenerative medicine. However, the availability and capacity for self-renewal are limited under current preparation protocols. iPSCs now provide an alternative, similar cell source for MSCs. Therefore, certain embodiments of this disclosure provide a method for differentiating MSCs from iPSCs (e.g., additionally reprogrammed iPSCs) by initiating mesodermal differentiation on a positively charged amine surface using GMP-compatible conditions. MSCs derived through this process express all purity markers of the MSC lineage and exhibit self-renewal and pluripotency. These cells can be scaled up for clinical applications.

[0101] In a further embodiment, this disclosure provides a method for differentiating MSCs into pericytes (PCs). Pericytes (also known as parietal cells) surround blood microvessels (i.e., capillaries, arterioles, and venules) and are generally understood to play an tissue or structural role in angiogenesis. Several criteria, including location, morphology, gene or protein expression patterns, and perivascular density, are used to identify immature and mature pericytes. Generally, pericytes obtained according to the methods provided herein can be identified based on the expression of known pericyte molecular markers such as, but not limited to, PDGFRβ, desmin (DES), CD13 (ANPEP; alanyl-1, α-SMA), RGS5 (G protein signaling regulator 5), NG2 (also known as CSPG4; chondroitin sulfate proteoglycan 4), CD248 (endothelial sialic acid protein), ANG-1, CD146, CD44, CD90, and CD13.

[0102] Microglia are innate immune cells of the central nervous system, playing a crucial role in brain development, homeostasis, and immune regulation. They are difficult to obtain from human fetuses and neonatal tissues. Therefore, further embodiments of this disclosure provide methods for generating, characterizing, and cryopreserving human iPSC-derived microglia (iMGLs) from HPCs (e.g., additionally reprogrammed iCell HPCs) under defined conditions. Cryopreserved iMGLs retain purity, secrete immunomodulatory cytokines, and phagocytose pHrodo Red-labeled bacterial bioparticles and amyloid β aggregates. The ability to generate a virtually unlimited number of iMGLs offers tremendous promise for accelerating human neuroscience research into the role of microglia in normal and disease states.

[0103] This article further presents microglia with disruptions in TREM2, MeCP2, and / or SCNA. These microglia derived from patient-derived iPSCs provide an in vitro tool for creating more accurate models to understand the complex interactions between human microglia, neurons, and astrocytes in 2D or 3D organoid systems and to mimic neuronal diseases.

[0104] This article also provides a method for differentiating iPSCs into neural progenitor cells (NPCs) without inhibiting SMAD signaling. These NPCs can be co-cultured with iPSC-derived microglia to generate long-term co-culture assays that mimic human brain development and the complex intercellular interactions between neural lineages, microglia, endothelial cells, pericytes, and astrocytes in discs derived from normal and / or disease-specific iPSCs. iPSCs can be maintained under hypoxic conditions to generate NPCs prior to differentiation initiation. To initiate neural progenitor differentiation, iPSCs can be plated on ECM-coated surfaces in the presence of a ROCK inhibitor or blebbistatin. Cells can then be placed in culture medium for the next 48 hours in the absence of a ROCK inhibitor. Subsequently, cells are pretreated for 72 hours in DMEMF12 medium supplemented with a GSK3 inhibitor, with the medium changed daily under normoxic conditions. Cells can be harvested at the end of the pretreatment step and then replate back onto ECM-coated plates in a 2D format, or replate as 3D aggregates using ultra-low adhesion plates or spinner flasks in the presence of a ROCK inhibitor or blebbistatin. Over the next 8 days, under normoxic conditions, the culture can be fed every other day with E6 medium containing N2 to produce NPCs. Figure 45A The document outlines the different steps involved in generating NPCs.

[0105] The cells generated by this method can be used for disease modeling, drug discovery, and regenerative medicine. This document also provides methods for generating brain organoids or blood-brain barrier (BBB) ​​models using the cells of this invention (e.g., MSCs, endothelial cells, neural progenitor cells, and pericytes).

[0106] I. Definition

[0107] As used herein, “a” or “an” may mean one or more. As used herein in the claims, when used in conjunction with the word “comprising”, the word “a” or “an” may mean one or more, or one or more kinds.

[0108] Unless explicitly stated otherwise, referring only to alternatives or that alternatives are mutually exclusive (however, this disclosure supports the definition of referring only to alternatives and "and / or"), the term "or" as used in the claims is used to mean "and / or". As used herein, "another" may mean at least a second or more.

[0109] The term “substantially” should be understood as meaning that a method or composition includes only the specified steps or materials, and those steps or materials that do not substantially affect the essential and novel features of those methods and compositions.

[0110] As used herein, compositions or culture media that are “substantially free” of the specified substance or material contain ≤30%, ≤20%, ≤15%, more preferably ≤10%, even more preferably ≤5%, or most preferably ≤1% of the substance or material.

[0111] As used herein, the terms “generally” or “approximately” may be used to modify any quantitative comparison, value, measurement or other representation that is permissible to vary without causing a change in the essential function associated with it.

[0112] The term "about" typically refers to the standard deviation of the value as determined using standard analytical techniques for measuring the value. These terms can also refer to adding or subtracting 5% from the value.

[0113] As used herein, "substantially free" with respect to a specified component is used to mean that no specified component is intentionally formulated into the composition and / or present only as a contaminant or in trace amounts. Therefore, the total amount of the specified component due to any unintentional contamination of the composition is well below 0.05%, preferably below 0.01%. Most preferably, a composition is one in which the amount of the specified component is undetectable by standard analytical methods.

[0114] "Feeder-free" or "feeder-independent" in this document refers to cultures supplemented with cytokines and growth factors (e.g., TGFβ, bFGF, LIF) as a substitute for a feeder cell layer. Therefore, feeder-free or feeder-independent culture systems and media can be used to culture and maintain pluripotent cells in an undifferentiated and proliferating state. In some cases, feeder-free cultures use animal-based substrates (e.g., MATRIGEL). TM They can be grown on substrates such as fibronectin, collagen, or fibronectin. These methods allow human stem cells to remain essentially undifferentiated without the need for a mouse fibroblast "feeder layer".

[0115] In this paper, "feeder layer" is defined as a coating of cells, such as at the bottom of a culture dish. Feeder cells are able to release nutrients into the culture medium and provide a surface on which other cells, such as pluripotent stem cells, can attach.

[0116] The terms "compositionally defined" or "fully defined," when used in relation to a culture medium, extracellular matrix, or culture condition, refer to a culture medium, extracellular matrix, or culture condition in which the chemical components and the amounts of virtually all components are known. For example, a compositionally defined medium does not contain indeterminate factors such as fetal bovine serum, bovine serum albumin, or human serum albumin. Typically, compositionally defined media include a basal medium (such as Duchenne Modified Eagle Medium (DMEM), F12, or Roswell Park Memorial Institute Medium (RPMI) 1640, containing amino acids, vitamins, inorganic salts, buffers, antioxidants, and energy) supplemented with recombinant albumin, chemically defined lipids, and recombinant insulin. An example of a fully defined medium is Essential 8. TM Culture medium.

[0117] For culture media, extracellular matrix, or culture systems used with human cells, the term "xenogeneic (XF)" means that the materials used therein are not derived from non-human animals.

[0118] "Treatment" or "cure" includes (1) suppressing the disease of a subject or patient experiencing or exhibiting the pathology or symptomology of the disease (e.g., preventing further development of the pathology and / or symptomology), (2) improving the disease of a subject or patient experiencing or exhibiting the pathology or symptomology of the disease (e.g., reversing the pathology and / or symptomology), and / or (3) achieving any measurable reduction in the disease of a subject or patient experiencing or exhibiting the pathology or symptomology of the disease.

[0119] "Preventive treatment" includes: (1) reducing or mitigating the risk of developing disease in any or all pathologies or symptomatologies of a subject or patient who may be at risk of and / or susceptible to disease but has not yet experienced or exhibited disease, and / or (2) slowing the onset of pathology or symptomatology of disease in any or all pathologies or symptomatologies of a subject or patient who may be at risk of and / or susceptible to disease but has not yet experienced or exhibited disease.

[0120] As used herein, the terms "patient" or "subject" refer to a living mammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat, mouse, rat, guinea pig, or a transgenic species thereof. In some embodiments, the patient or subject is a primate. Non-limiting examples of human patients include adults, adolescents, infants, and fetuses.

[0121] The term "effective" as used in this specification and / or claims means sufficient to achieve the desired, anticipated, or intended result. When used in the context of treating a patient or subject with a compound, "effective amount," "therapeutic effective amount," or "pharmaceutical effective amount" means an amount sufficient to affect the disease when administered to a subject or patient for the treatment or prevention of the disease.

[0122] As used generally in this article, "pharmaceutically acceptable" means those compounds, materials, compositions, and / or dosage forms that, to a reasonable extent of medical judgment, are suitable for use in contact with the tissues, organs, and / or body fluids of humans and animals without excessive toxicity, irritation, allergic reactions, or other problems or complications commensurate with a reasonable benefit / risk ratio.

[0123] "Induced pluripotent stem cells (iPSCs)" are cells generated by reprogramming somatic cells through the expression or induction of a combination of expression factors (referred to herein as reprogramming factors). iPSCs can be generated from fetal, postpartum, neonatal, adolescent, or adult somatic cells. In some embodiments, factors that can be used to reprogram somatic cells into pluripotent stem cells include, for example, Oct4 (sometimes referred to as Oct3 / 4), Sox2, c-Myc, Klf4, Nanog, and Lin28. In some embodiments, somatic cells are reprogrammed into pluripotent stem cells by expressing at least two, at least three, or four reprogramming factors.

[0124] The term "extracellular matrix protein" refers to molecules that provide structural and biochemical support to surrounding cells. Extracellular matrix proteins can be recombinant and can also refer to fragments or peptides. Examples include collagen and heparin sulfate.

[0125] "Three-dimensional (3-D) culture" refers to an artificially created environment in which biological cells are allowed to grow or interact with their surroundings in all three dimensions. 3-D cultures can be grown in a variety of cell culture containers, such as bioreactors, small capsules in which cells can grow into spherical shapes, or non-adherent culture plates. In a specific respect, 3-D culture is scaffold-free. Conversely, "two-dimensional (2-D)" culture refers to cell cultures on adherent surfaces, such as monolayers.

[0126] As used herein, “destruction” of a gene refers to the elimination or reduction of expression of one or more gene products encoded by a subject gene in a cell, compared to the expression level of the gene product in the absence of destruction. Exemplary gene products include gene-encoded mRNA and protein products. In some cases, destruction is temporary or reversible, while in others it is permanent. In some cases, destruction is the disruption of a functional or full-length protein or mRNA, although truncated or nonfunctional products may indeed result. In some embodiments herein, gene activity or function is disrupted, rather than expression. Gene destruction is typically induced by artificial methods, i.e., by adding or introducing compounds, molecules, complexes, or compositions, and / or by disrupting the nucleic acids of a gene or gene-associated nucleic acids, such as at the DNA level. Exemplary methods of gene destruction include gene silencing, knockdown, knockout, and / or gene destruction techniques, such as gene editing. Examples include antisense techniques, such as RNAi, siRNA, shRNA, and / or ribozymes, which typically result in a transient reduction in expression, and gene editing techniques that result in the inactivation or destruction of a targeted gene, such as by inducing breakage and / or homologous recombination. Examples include insertions, mutations, and deletions. Disruptions typically result in the suppression and / or complete loss of expression of the normal or "wild-type" product encoded by the gene. Instances of such gene disruption are insertions, frameshifts, and missense mutations, deletions, knock-ins, and knockouts of a gene or part of a gene, including the deletion of the entire gene. Such disruptions can occur in coding regions, for example, in one or more exons, resulting in the inability to produce a full-length product, a functional product, or any product, for example, by inserting a stop codon. Such disruptions can also occur through disruption of promoters or enhancers or other regions affecting transcriptional activation, thereby preventing gene transcription. Gene disruption includes gene targeting, including the inactivation of targeted genes through homologous recombination.

[0127] II. iPSC Differentiation Methods

[0128] A.HPC

[0129] iPSCs can be differentiated into HPCs using methods known in the art, such as those described in U.S. Patent No. 8,372,642, which is incorporated herein by reference. In one method, a combination of BMP4, VEGF, Flt3 ligand, IL-3, and GM-CSF can be used to promote hematopoietic differentiation. In some embodiments, cell cultures are sequentially exposed to a first medium to prepare iPSCs for differentiation, a second medium containing BMP4, VEGF, and FGF, and then cultured in a third medium containing Flt3 ligand, SCF, TPO, IL-3, and IL-6, which can differentiate pluripotent cells into HPCs and hematopoietic cells. The second component-determining medium may also contain heparin. Furthermore, the addition of FGF-2 (50 ng / ml) to a medium containing BMP4 and VEGF can improve the efficiency of generating hematopoietic progenitor cells from pluripotent cells. In addition, the addition of a glycogen synthase kinase 3 (GSK3) inhibitor (e.g., CHIR99021, BIO, and SB-216763) to the first component-determining medium can further enhance HPC production.

[0130] Typically, pluripotent cells can be differentiated into hematopoietic progenitor cells using defined or indeterminate conditions. It should be understood that defined conditions are generally preferred in embodiments where the resulting cells are intended for use in human subjects. Hematopoietic stem cells can be derived from pluripotent stem cells under defined conditions (e.g., using TeSR medium), and hematopoietic cells can be generated from germ-like structures derived from pluripotent cells. In other embodiments, pluripotent cells can be co-cultured on OP9 cells or mouse embryonic fibroblasts and subsequently differentiated.

[0131] As part of the differentiation process, pluripotent cells can be allowed to form embryoid bodies or aggregates. The formation of "embryoid bodies" (EBs), or clusters of growing cells, to induce differentiation typically involves the in vitro aggregation of human pluripotent stem cells into EBs and allowing them to spontaneously and randomly differentiate into multiple tissue types representing endoderm, ectoderm, and mesoderm origins. Therefore, three-dimensional EBs can be used to generate a proportion of hematopoietic cells and endothelial cells.

[0132] To promote aggregate formation, cells were transferred to low-attachment plates for overnight incubation in serum-free differentiation (SFD) medium consisting of 75% IMDM (Gibco), 25% Ham's Modified F12 (Cellgro) supplemented with 0.05% N2 and 1% B-27 without RA supplementation, 200 mM 1-glutamine, 0.05 mg / ml magnesium ascorbate-2-phosphate (Asc 2-P) (WAKO), and 4.5 x 10⁻⁶ ppm. -4The medium consists of MTG. Cells can be collected from each well and centrifuged on the second day. For the first four days of differentiation, cells are then resuspended in "EB differentiation medium," which is a basal SFD medium supplemented with approximately 50 ng / ml bone morphogenetic factor (BMP4), approximately 50 ng / ml vascular endothelial growth factor (VEGF), and 50 ng / ml zb FGF. Cells are semi-feeded every 48 hours. On the fifth day of differentiation, the medium is replaced with a second medium consisting of SFD medium supplemented with 50 ng / ml stem cell factor (SCF), approximately 50 ng / ml Flt-3 ligand (Flt-3L), 50 ng / ml interleukin-6 (IL-6), 50 ng / ml interleukin-3 (IL-3), and 50 ng / ml thrombopoietin (TPO). Cells are semi-feeded every 48 hours with fresh differentiation medium. Medium replacement is performed by centrifuging the differentiation culture at 300g for 5 minutes, aspirating half the volume of the differentiation culture, and replenishing it with fresh medium. In some embodiments, the EB differentiation medium may include approximately BMP4 (e.g., approximately 50 ng / ml), VEGF (e.g., approximately 50 ng / ml), and optionally FGF-2 (e.g., approximately 25-75 ng / ml or approximately 50 ng / ml). The supernatant can be aspirated and replaced with fresh differentiation medium. Alternatively, cells can be fed semi-feeded with fresh medium every two days. Cells can be harvested at different time points during the differentiation process.

[0133] HPCs can be cultured from pluripotent stem cells using a defined culture medium. Pluripotent cells can then be differentiated into hematopoietic CD34 cells using this defined culture medium. + Stem cell methods are described, for example, in U.S. Patent Application 12 / 715,136, the entire contents of which are incorporated herein by reference. These methods are intended to be used in this disclosure.

[0134] For example, a culture medium with defined components can be used to induce hematopoietic CD34. + Differentiation. The composition-defined culture medium may contain growth factors BMP4, VEGF, Flt3 ligand, IL-3, and / or GMCSF. Pluripotent cells can be cultured in a first composition-defined medium containing BMP4, VEGF, and optionally FGF-2, followed by a second medium containing (Flt3 ligand, IL-3, and GMCSF) or (Flt3 ligand, IL-3, IL-6, and TPO). The first and second media may also contain one or more of SCF, IL-6, G-CSF, EPO, FGF-2, and / or TPO. Essentially hypoxic conditions (e.g., below 20% O2) may further promote hematopoiesis or endothelial differentiation.

[0135] Cells can be activated by mechanical or enzymatic tools (e.g., using trypsin or TrypLE).TM This is essentially individualized. ROCK inhibitors (such as H1152 or Y-27632) can also be included in the culture medium. These methods are expected to be automated, for example, using robotics.

[0136] In some embodiments, substantially hypoxic conditions can be used to promote the differentiation of pluripotent cells into hematopoietic progenitor cells. Those skilled in the art will recognize that an atmospheric oxygen content of less than about 20.8% is considered hypoxic. Cultured human cells are capable of growing under atmospheric conditions with reduced oxygen content compared to ambient air. This relative hypoxia can be achieved by reducing exposure to atmospheric oxygen in the culture medium. Embryonic cells typically develop in vivo under reduced oxygen conditions (typically between about 1% and about 6% atmospheric oxygen) with carbon dioxide at ambient levels. Not wishing to be bound by theory, it is anticipated that hypoxic conditions can mimic one aspect of certain embryonic developmental conditions. As illustrated in the following examples, hypoxic conditions can be used in some embodiments to promote the further differentiation of induced pluripotent cells into more differentiated cell types, such as HPC.

[0137] The following hypoxic conditions can be used to promote the differentiation of pluripotent cells into hematopoietic progenitor cells. In some embodiments, atmospheric oxygen content of less than about 20%, less than about 19%, less than about 18%, less than about 17%, less than about 16%, less than about 15%, less than about 14%, less than about 13%, less than about 12%, less than about 11%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, about 5%, about 4%, about 3%, about 2%, or about 1% can be used to promote differentiation into hematopoietic precursor cells. In some embodiments, the hypoxic atmosphere contains about 5% oxygen.

[0138] Regardless of the specific culture medium used in any given hematopoietic progenitor cell expansion, the medium is preferably supplemented with at least one cytokine at a concentration of about 0.1 ng / mL to about 500 ng / mL, more typically 10 ng / mL to 100 ng / mL. Suitable cytokines include, but are not limited to, c-kit ligand (KL) (also known as gray factor (StI), mast cell growth factor (MGF), and stem cell factor (SCF)), IL-6, G-CSF, IL-3, GM-CSF, IL-1α, IL-11MIP-1α, LIF, c-mpl ligand / TPO, and flk2 / flk3 ligand (Flt2L or Flt3L). In particular, the culture will contain at least one of SCF, Flt3L, and TPO. More specifically, the culture will contain SCF, Flt3L, and TPO.

[0139] In one implementation, cytokines are contained in a culture medium and replenished via perfusion. Alternatively, when using a bioreactor system, cytokines can be added separately as a concentrated solution through a separate inlet without perfusion. When cytokines are added without perfusion, they are typically added as a 10x to 100x solution in an amount equal to one-tenth to one-hundredth of the bioreactor volume, and fresh cytokines are added approximately every 2 to 4 days. Furthermore, in addition to the cytokines perfused in the culture medium, freshly concentrated cytokines can also be added separately.

[0140] Exemplary HPC Differentiation Method

[0141] 2D HPC Differentiation: iPSC can be maintained in the presence of E8 at MATRIGEL TM Cells were either placed on blebbistatin and adapted to hypoxia for at least 5-10 passages. Cells were separated from sub-merging iPSCs and plated onto amine plates at a density of 250,000 cells / well in serum-free fractionally determined (SFD) medium supplemented with 5 μM blebbistatin. 24 hours after platening, SFD medium supplemented with 50 ng / ml BMP4, VEGF, and FGF2 was added to the culture. The next day, the medium was replaced with fresh medium to remove blebbistatin. On day 5 of differentiation, cells were placed in medium containing 50 ng / ml Flt-3 ligand, SCF, TPO, IL3, and IL6, and 5 U / ml heparin. Cells were fed every 48 hours throughout differentiation. The entire process was performed under hypoxic conditions on charged amine plates. HPC was quantified by the presence of CD43 / CD34 cells and CFU.

[0142] 3D HPC Differentiation: Cells were separated from sub-confluent iPSCs and seeded into spin flasks at a density of 250,000-500,000 cells / mL in serum-free (SFD) medium supplemented with 5 μM blebbistatin or 1 μM H1152. The medium was replaced with SFD supplemented with 50 ng / mL BMP4, VEGF, and FGF2 24 hours after seeding. On day 5 of differentiation, cells were placed in medium containing 50 ng / mL Flt-3 ligand, SCF, TPO, IL3, and IL6, and 5-10 U / mL heparin. Cells were fed every 48 hours throughout the differentiation process. The entire process was performed under hypoxic conditions. HPC was quantified by the presence of CD43 / CD34. HPC was sorted using CD34 beads via MACS.

[0143] B. Gene damage

[0144] In some respects, the expression, activity, or function of TREM2, MeCP2, and / or SCNA genes is disrupted in cells such as PSCs (e.g., ESCs or iPSCs). In some embodiments, gene disruption is achieved by inducing damage within the gene (e.g., knockout, insertion, missense, or frameshift mutations, such as biallelic frameshift mutations, deletion and / or knock-in of all or part of the gene (e.g., one or more exons or portions thereof)). For example, disruption can be achieved by sequence-specific or targeted nucleases (including DNA-binding targeted nucleases, such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), and RNA-guided nucleases, such as CRISPR-associated nucleases (Cases), specifically designed to target sequences or portions of the gene).

[0145] In some implementations, the disruption of gene expression, activity, and / or function is achieved by disrupting the gene. In some aspects, the gene is disrupted such that its expression is reduced by at least or about 20, 30, or 40%, typically at least or about 50, 60, 70, 80, 90, or 95%, compared to expression in the absence of gene disruption or the absence of the component introduced to achieve disruption.

[0146] In some implementations, the damage is transient or reversible, allowing gene expression to recover later. In other implementations, the damage is not reversible or temporary, for example, it is permanent.

[0147] In some embodiments, gene disruption is typically achieved in a targeted manner by inducing one or more double-strand breaks and / or one or more single-strand breaks in the gene. In some embodiments, the double-strand or single-strand breaks are generated by nucleases such as endonucleases or gene-targeting nucleases. In some aspects, breaks are induced in the coding region of the gene, such as in exons. For example, in some embodiments, induction occurs near the N-terminal portion of the coding region, such as in the first exon, the second exon, or subsequent exons.

[0148] In some respects, double-strand or single-strand breaks are repaired through cellular repair processes, such as non-homologous end joining (NHEJ) or homologous directed repair (HDR). In other respects, the repair process is prone to error and can lead to gene disruption, such as frameshift mutations, including biallelic frameshift mutations, which can result in complete gene knockout. For example, in some respects, disruption includes induced deletions, mutations, and / or insertions. In some embodiments, disruption results in the presence of an early stop codon. In some respects, insertions, deletions, translocations, frameshift mutations, and / or the presence of early stop codons lead to disruption of gene expression, activity, and / or function.

[0149] In some implementations, antisense techniques are used to achieve gene disruption, such as through RNA interference (RNAi), short interfering RNA (siRNA), short hairpin RNA (shRNA), and / or the use of ribozymes to selectively inhibit or repress gene expression. siRNA technology is RNAi that utilizes a double-stranded RNA molecule having a sequence homologous to the nucleotide sequence of the mRNA transcribed from the gene and a sequence complementary to the nucleotide sequence. siRNA is typically homologous / complementary to a region of the mRNA transcribed from the gene, or it can be a siRNA comprising multiple RNA molecules homologous / complementary to different regions. In some aspects, siRNA is contained in a polycistronic construct. In specific aspects, siRNA inhibits the translation of wild-type and mutant proteins from endogenous mRNA.

[0150] In some embodiments, disruption is achieved using DNA-targeting molecules, such as DNA-binding proteins or DNA-binding nucleic acids, or complexes, compounds, or compositions containing them, which specifically bind to or hybridize to genes. In some embodiments, the DNA-targeting molecule contains a DNA-binding domain, such as a zinc finger protein (ZFP) DNA-binding domain, a transcription activator-like protein (TAL) or TAL effector (TALE) DNA-binding domain, a clustered regularly spaced short palindromic repeat (CRISPR) DNA-binding domain, or a DNA-binding domain derived from a meganuclease. Zinc finger, TALE, and CRISPR system binding domains can be engineered to bind predetermined nucleotide sequences, for example, by engineering (altering one or more amino acids) the recognition helical region of naturally occurring zinc finger or TALE proteins. Engineered DNA-binding proteins (zinc fingers or TALEs) are non-naturally occurring proteins. Reasonable design criteria include the application of substitution rules and computer algorithms to process information in databases storing existing ZFP and / or TALE designs and binding data. See, for example, U.S. Patent Nos. 6,140,081; 6,453,242; and 6,534,261; also see WO98 / 53058; WO98 / 53059; WO98 / 53060; WO02 / 016536 and WO03 / 016496 and U.S. Publication No. 2011 / 0301073.

[0151] In some embodiments, the DNA-targeting molecule, complex, or combination comprises a DNA-binding molecule and one or more additional domains, such as effector domains that promote gene repression or disruption. For example, in some embodiments, gene disruption is carried out via a fusion protein comprising a DNA-binding protein and a heterologous regulatory domain or a functional fragment thereof. In some aspects, the domains include, for example, transcription factor domains, such as activators, repressors, coactivators, co-repressors, silencers, oncogenes, DNA repair enzymes and their associated factors and modifiers, DNA rearrangement enzymes and their associated factors and modifiers, chromatin-associated proteins and their modifiers, such as kinases, acetyltransferases, and deacetylases, and DNA-modifying enzymes, such as methyltransferases, topoisomerases, helicases, ligases, kinases, phosphatases, polymerases, endonucleases, and their associated factors and modifiers. See, for example, U.S. Patent Application Publications 2005 / 0064474; 2006 / 0188987 and 2007 / 0218528, which are incorporated herein by reference in their entirety, providing details of fusions of DNA-binding domains and nuclease-cutting domains. In some aspects, the additional domain is a nuclease domain structure. Thus, in some embodiments, gene disruption facilitated by gene or genome editing is achieved using engineered proteins, such as nucleases and nuclease-containing complexes or fusion proteins, comprising sequence-specific DNA-binding domains fused or complexed with nonspecific DNA-cutting molecules such as nucleases.

[0152] In some respects, these targeted chimeric nucleases or nuclease-containing complexes perform precise genetic modifications by inducing targeted double-strand breaks or single-strand breaks and stimulating cellular DNA repair mechanisms, including error-prone non-homologous end joining (NHEJ) and homologous directed repair (HDR). In some embodiments, the nuclease is an endonuclease, such as a zinc finger nuclease (ZFN), a TALE nuclease (TALEN), or an RNA-guided endonuclease (RGEN), such as a CRISPR-associated (Cas) protein or a meganuclease.

[0153] In some embodiments, donor nucleic acids, such as donor plasmids or nucleic acids encoding genetically engineered antigen receptors, are provided and inserted into gene editing sites via HDR after introduction into the DSB. Thus, in some embodiments, gene disruption and the introduction of antigen receptors, such as CARs, occur simultaneously, thereby partially disrupting the gene by knocking in or inserting nucleic acids encoding CARs.

[0154] In some implementations, donor nucleic acids are not provided. In some aspects, NHEJ-mediated repair following the introduction of DSB results in insertion or deletion mutations, which can lead to gene damage, for example, by generating missense mutations or frameshifts.

[0155] 1. ZFP and ZFN

[0156] In some implementations, the DNA-targeting molecule includes a DNA-binding protein fused to an effector protein, such as an endonuclease, such as one or more zinc finger proteins (ZFPs) or transcription activator-like proteins (TALs). Examples include ZFN, TALE, and TALEN.

[0157] In some implementations, the DNA-targeting molecule contains one or more zinc finger proteins (ZFPs) or their domains that bind to DNA in a sequence-specific manner. A ZFP or its domain is a protein or domain within a larger protein that binds to DNA in a sequence-specific manner via one or more zinc fingers (amino acid sequence regions within the binding domain whose structure is stabilized by coordination with zinc ions). The term zinc finger DNA-binding protein is often abbreviated as zinc finger protein or ZFP. Artificial ZFP domains, typically 9-18 nucleotides in length, are generated from the assembly of individual fingers and target specific DNA sequences.

[0158] ZFPs include those in which a single finger domain is approximately 30 amino acids long and contains an α-helix containing two invariant histidine residues coordinated to two cysteine ​​residues via a single β-turn, and having two, three, four, five, or six fingers. Typically, the sequence specificity of ZFPs can be altered by amino acid substitutions at four helical positions (-1, 2, 3, and 6) on the zinc finger recognition helix. Therefore, in some embodiments, ZFPs or ZFP-containing molecules are not naturally occurring, for example, engineered to bind to selected target sites.

[0159] In some respects, MeCP2 disruption is achieved by contacting a first target site in the gene with a first ZFP, thereby disrupting the gene. In some embodiments, the target site in the gene contacts a fusion ZFP containing six fingers and regulatory domains, thereby inhibiting gene expression.

[0160] In some embodiments, the contacting step further includes contacting the second target site in the gene with the second ZFP. In some aspects, the first and second target sites are adjacent. In some embodiments, the first and second ZFPs are covalently linked. In some aspects, the first ZFP is a fusion protein containing one or at least two regulatory domains.

[0161] In some embodiments, the first and second ZFPs are fusion proteins, each containing one regulatory domain or each containing at least two regulatory domains. In some embodiments, the regulatory domains are transcriptional repressors, transcriptional activators, endonucleases, methyltransferases, histone acetyltransferases, or histone deacetylases.

[0162] In some embodiments, the ZFP is encoded by a ZFP nucleic acid operatively linked to a promoter. In some aspects, the method further includes the step of first administering the nucleic acid to cells in a lipid:nucleic acid complex or as a naked nucleic acid. In some embodiments, the ZFP is encoded by an expression vector containing a ZFP nucleic acid operatively linked to a promoter. In some embodiments, the ZFP is encoded by a nucleic acid operatively linked to an inducible promoter. In some aspects, the ZFP is encoded by a nucleic acid operatively linked to a weak promoter.

[0163] In some implementations, the target site is upstream of the transcription start site of the gene. In some aspects, the target site is adjacent to the transcription start site of the gene. In some aspects, the target site is adjacent to an RNA polymerase pause site downstream of the transcription start site of the gene.

[0164] In some embodiments, the molecule targeting DNA is or contains a zinc finger DNA-binding domain fused to a DNA cleavage domain to form a zinc finger nuclease (ZFN). In some embodiments, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one liS-type restriction enzyme and one or more zinc finger binding domains, which may or may not be engineered. In some embodiments, the cleavage domain is derived from the liS-type restriction endonuclease FokI. FokI typically catalyzes double-stranded cleavage of DNA at 9 nucleotides from its recognition site on one strand and 13 nucleotides from its recognition site on the other strand.

[0165] In some embodiments, ZFN targets genes present in engineered cells. In some aspects, ZFN efficiently generates double-strand breaks (DSBs), for example, at predetermined sites within gene coding regions. Typical targeted regions include exons, regions coding N-terminal regions, first exons, second exons, and promoter or enhancer regions. In some embodiments, transient expression of ZFN promotes efficient and permanent disruption of target genes in engineered cells. Specifically, in some embodiments, ZFN delivery results in permanent gene disruption with an efficiency exceeding 50%.

[0166] Many gene-specific engineered zinc fingers are commercially available. For example, Sangamo Biosciences (Richmond, CA, USA) collaborated with Sigma-Aldrich (St. Louis, MO, USA) to develop a platform (CompoZr) for zinc finger construction, enabling researchers to completely bypass zinc finger construction and validation and provide specific targeting zinc fingers for thousands of proteins (Gaj et al., Trends in Biotechnology, 2013, 31(7), 397-405). In some implementations, commercially available zinc fingers are used or custom-designed.

[0167] 2. TAL, TALE, and TALEN

[0168] In some embodiments, the DNA-targeting molecule comprises a naturally occurring or engineered (non-naturally occurring) transcription activator-like protein (TAL) DNA-binding domain, such as in transcription activator-like protein effector (TALE) proteins, see, for example, U.S. Patent Publication No. 2011 / 0301073, the entire contents of which are incorporated herein by reference.

[0169] A TALE DNA-binding domain, or TALE, is a polypeptide containing one or more TALE repeat domains / units. The repeat domains are involved in the binding of the TALE to its homologous target DNA sequence. A single “repeat unit” (also called a “repeat sequence”) is typically 33-35 amino acids in length and shows at least some sequence homology with other TALE repeat sequences within naturally occurring TALE proteins. Each TALE repeat unit contains one or two DNA-binding residues, constituting a repeat variable double residue (RVD), typically located at positions 12 and / or 13 of the repeat sequence. Natural (typical) codes for DNA recognition of these TALEs have been determined, such that HD sequences at positions 12 and 13 result in binding to cytosine (C), NG to T, NI to A, NN to G or A, and NO to T, and non-classical (atypical) RVDs are also known. See U.S. Patent Publication No. 2011 / 0301073. In some embodiments, by designing TAL arrays specific to the target DNA sequence, TALEs can target any gene. Target sequences typically begin with thymidine.

[0170] In some implementations, the molecule is a DNA-binding endonuclease, such as TALE nuclease (TALEN). In some aspects, TALEN is a fusion protein comprising a DNA-binding domain derived from TALE and a nuclease catalytic domain that cleaves a target nucleic acid sequence.

[0171] In some embodiments, TALEN recognizes and cleaves target sequences in a gene. In some aspects, DNA cleavage results in double-strand breaks. In some aspects, the breaks stimulate the rate of homologous recombination or non-homologous end joining (NHEJ). Typically, NHEJ is an imperfect repair process that usually results in changes to the DNA sequence at the cleavage site. In some aspects, the repair mechanism involves rejoining the remaining portions of the two DNA ends by direct rejoining (Critchlow and Jackson, 1998) or by so-called microhomologous-mediated end joining. In some embodiments, repair via NHEJ results in small insertions or deletions and can be used to disrupt and thereby repress the gene. In some embodiments, the modification can be a substitution, deletion, or addition of at least one nucleotide. In some aspects, cells in which cleavage-induced mutagenesis events, i.e., mutagenesis events consecutive to NHEJ events, can be identified and / or selected by methods well known in the art.

[0172] In some implementations, TALE repeat sequences are assembled to specifically target genes. TALEN libraries targeting 18,740 individual protein-coding genes have been constructed. Custom-designed TALE arrays are commercially available from Cellectis Bioresearch (Paris, France), Transposagen Biopharmaceuticals (Lexington, KY, USA), and Life Technologies (Grand Island, NY, USA).

[0173] In some implementations, TALEN is introduced as a transgene encoded by one or more plasmid vectors. In some aspects, the plasmid vector may contain selection markers that provide for the identification and / or selection of cells receiving the vector.

[0174] 3. RGEN (CRISPR / Cas system)

[0175] In some implementations, disruption is performed using one or more DNA-binding nucleic acids, such as disruption via RNA-guided endonucleases (RGENs). For example, disruption can be performed using clusters of regularly spaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins. Generally, the “CRISPR system” refers to transcripts and other elements involved in the expression of CRISPR-associated (“Cas”) genes or directing their activity, including sequences encoding Cas genes, tracr (trans-activating CRISPR) sequences (e.g., tracrRNA or active portion of tracrRNA), tracr pairing sequences (including “directed repeat sequences” and partially directed repeat sequences processed by tracrRNA in the context of the endogenous CRISPR system), guide sequences (also referred to as “spacer regions” in the context of the endogenous CRISPR system), and / or other sequences and transcripts from CRISPR loci.

[0176] CRISPR / Cas nucleases or CRISPR / Cas nuclease systems may include a non-coding RNA molecule (guide) RNA (which binds to DNA specifically) and a Cas protein (e.g., Cas9) having nuclease function (e.g., two nuclease domains). One or more elements of a CRISPR system may be derived from a type I, type II, or type III CRISPR system, for example from a specific organism containing an endogenous CRISPR system, such as Streptococcus pyogenes.

[0177] In some cases, Cas nucleases and gRNAs (including fusions of target sequence-specific crRNAs and fixed tracrRNAs) are introduced into cells. Typically, the 5' target site of the gRNA targets the Cas nuclease to a target site, such as a gene, using complementary base pairing. The target site can be selected based on the 5' position of its immediate neighboring motif (PAM) sequence (e.g., typically NGG or NAG). In this regard, the gRNA targets the desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. Generally, CRISPR systems are characterized by elements that promote the formation of a CRISPR complex at the target sequence site. Typically, the "target sequence" usually refers to a sequence to which the guide sequence is designed to be complementary, where hybridization between the target and guide sequences promotes the formation of the CRISPR complex. Perfect complementarity is not required, as long as there is sufficient complementarity to induce hybridization and promote the formation of the CRISPR complex.

[0178] The CRISPR system can induce double-strand breaks (DSBs) at the target site, followed by disruption as discussed herein. In other embodiments, a Cas9 variant, considered a “nicking enzyme,” is used to cleave a single strand at the target site. Paired nicking enzymes can be used, for example, to improve specificity, with each nicking enzyme guided by a pair of different gRNA targeting sequences, such that a 5' overhang is introduced simultaneously upon introduction of the nick. In other embodiments, catalytically inactivated Cas9 is fused with a heterologous effector domain, such as a transcriptional repressor or activator, to influence gene expression.

[0179] The target sequence can contain any polynucleotide, such as DNA or RNA polynucleotides. The target sequence can be located in the cell nucleus or cytoplasm, for example, within a cell organelle. Typically, the sequence or template that can be used for recombination into a target locus containing the target sequence is called the "edit template," "edit polynucleotide," or "edit sequence." In some respects, the exogenous template polynucleotide can be called the edit template. In some respects, recombination is homologous recombination.

[0180] Typically, in the context of an endogenous CRISPR system, the formation of a CRISPR complex (containing a guide sequence that hybridizes to the target sequence and is complexed with one or more Cas proteins) results in the cleavage of one or both strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more base pairs from the target sequence). A tracr sequence (which may contain all or part of the wild-type tracr sequence or consist of it (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85 or more nucleotides of the wild-type tracr sequence) may also form part of the CRISPR complex, for example, by hybridizing along at least a portion of the tracr sequence with all or part of a tracr pairing sequence operatively linked to the guide sequence. The tracr sequence and the tracr pairing sequence have sufficient complementarity to hybridize and participate in the formation of the CRISPR complex, for example, at least 50%, 60%, 70%, 80%, 90%, 95%, or 99% sequence complementarity along the length of the tracr pairing sequence at optimal alignment.

[0181] One or more vectors driving the expression of one or more elements of the CRISPR system can be introduced into cells such that the expression of the CRISPR system elements directs the formation of the CRISPR complex at one or more target sites. Components can also be delivered to cells as proteins and / or RNA. For example, the Cas enzyme, a guide sequence linked to a tracr-pairing sequence, and the tracr sequence can each be operatively linked to a separate regulatory element on a separate vector. Alternatively, two or more elements expressed from the same or different regulatory elements can be combined in a single vector, wherein one or more additional vectors provide any components of the CRISPR system not included in the first vector. The vector may contain one or more insertion sites, such as restriction endonuclease recognition sequences (also known as “cloning sites”). In some embodiments, one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When using multiple different guide sequences, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences within the cell.

[0182] The vector may contain regulatory elements operatively linked to an enzyme-coding sequence that encodes a CRISPR enzyme (e.g., the Cas protein). Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologues, or modified forms thereof. These enzymes are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database with accession number Q99ZW2.

[0183] The CRISPR enzyme can be Cas9 (e.g., derived from Streptococcus pyogenes or Streptococcus pneumoniae). The CRISPR enzyme can direct the cleavage of one or both strands at a location on the target sequence, such as within the target sequence and / or within a complementary sequence to the target sequence. The vector can encode a CRISPR enzyme mutated relative to the corresponding wild-type enzyme, such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of the target polynucleotide containing the target sequence. For example, the substitution of aspartic acid to alanine (D10A) in the RuvCI catalytic domain of Cas9 derived from Streptococcus pyogenes transforms Cas9 from a two-strand nuclease into a cleaving enzyme (single-strand cleavage). In some embodiments, the Cas9 cleaving enzyme can be used in combination with a guide sequence (e.g., two guide sequences, each targeting the sense and antisense strands of the DNA target, respectively). This combination allows both strands to be cleaved and used to induce NHEJ or HDR.

[0184] In some implementations, the enzyme-coding sequence encoding a CRISPR enzyme is codon-optimized for expression in specific cells, such as eukaryotic cells. Eukaryotic cells can be specific organisms (e.g., mammals, including but not limited to humans, mice, rats, rabbits, dogs, or non-human primates) or eukaryotic cells derived from them. Generally, codon optimization refers to the process of modifying a nucleic acid sequence to enhance expression in the host cell of interest while preserving the native amino acid sequence by replacing at least one codon of the native sequence with a codon that is more frequently or most frequently used in the host gene. Various species exhibit specific codon biases for certain amino acids. Codon bias (differences in codon use between organisms) is generally associated with the translation efficiency of messenger RNA (mRNA), which is considered to depend, among other things, on the characteristics of the codons being translated and the availability of specific transfer RNA (tRNA) molecules. The dominance of the selected tRNA in a cell generally reflects the most frequently used codons in peptide synthesis. Therefore, genes can be tailored based on codon optimization to achieve optimal gene expression in a given organism.

[0185] Generally, a guide sequence is any polynucleotide sequence that is sufficiently complementary to the target polynucleotide sequence to hybridize with the target sequence and guide the CRISPR complex to bind sequence-specifically to the target sequence. In some embodiments, when optimally aligned using a suitable alignment algorithm, the complementarity between the guide sequence and its corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.

[0186] The best alignment can be determined using any suitable algorithm for the alignment sequence. Non-limiting examples include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler transform (e.g., Burrows-Wheeler Aligner), Clustal W, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0187] CRISPR enzymes can be part of a fusion protein containing one or more heterologous protein domains. CRISPR enzyme fusion proteins can contain any additional protein sequence and optionally include a linker sequence between any two domains. Examples of protein domains that can be fused to a CRISPR enzyme include, but are not limited to, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methyltransferase activity, demethyltransferase activity, transcriptional activation activity, transcriptional repression activity, transcriptional release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-5-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins, including blue fluorescent protein (BFP). CRISPR enzymes can be fused with gene sequences encoding proteins or protein fragments that bind to DNA molecules or other cellular molecules, including, but not limited to, maltose-binding protein (MBP), S-tags, Lex A DNA-binding domain (DBD) fusions, GAL4A DNA-binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions.

[0188] C. Charged cell surface

[0189] In some embodiments, this disclosure relates to charged surfaces for cell culture. Charged surfaces can be positively charged, such as amine surfaces or nitrogen-containing functional groups, or negatively charged, such as carboxyl surfaces or oxygen-containing functional groups. Cell surfaces can be treated to alter the surface charge of the culture vessel.

[0190] In some respects, surfaces are neutrally charged, such as surfaces containing both negatively and positively charged functional groups. For example, CORNING... The surface possesses a unique mixture of oxygen-containing (negatively charged) and nitrogen-containing (positively charged) functional groups on the polystyrene surface. When cultured on a conventional TC surface, the surface supports cell growth, which may exhibit poor adhesion or limited differentiation potential. In some respects, the surface includes a ULA surface coating. For example, Corning's ultra-low adhesion surface is a covalently bonded hydrogel layer that is hydrophilic and neutrally charged. As proteins and other biomolecules are passively adsorbed onto the polystyrene surface through hydrophobic or ionic interactions, this hydrogel naturally inhibits nonspecific fixation by these forces, thereby suppressing subsequent cell adhesion. This surface is highly stable, non-cytotoxic, bioinert, and non-degradable. Other examples that can support the generation of microglia from HPC include Corning CellBIND culture (US Patent 6,617,152), which uses higher-energy microwave plasma to incorporate more oxygen into the polystyrene surface, making it more hydrophilic (wettable) than surfaces treated with conventional plasma or corona discharge, while also increasing surface stability. CorningSynthemax self-coating substrate is a unique, animal-free, viscosin-based synthetic peptide containing the RGD motif and flanking sequences. This synthetic peptide is covalently bound to the polymer backbone for passive coating, orientation, and presentation of the peptide for optimal cell binding and signal transduction.

[0191] Cell culture surfaces can be coated with a plasma-polymerized membrane. The source of plasma polymerization is one or more monomers. Useful polymerizable monomers may include unsaturated organic compounds such as enamines, haloalkenes, olefinic acids and carboxylates, nitriles, oxidized olefins, and olefins. In some embodiments, olefins may include vinyl and allyl forms. In other embodiments, cyclic compounds such as cyclohexane, cyclopentane, and cyclopropane may be used.

[0192] Those skilled in the art will recognize that various plasma polymerization techniques can be used to deposit one or more monomers onto cell culture surfaces. Preferably, a positively charged polymer film is deposited on the surface. As those skilled in the art will understand, depending on the protein used with it, the plasma-polymerized surface may have a negative charge. Amines are preferably used as the monomer source for the polymer. In some embodiments, the plasma-polymerized monomer is produced using a plasma source to generate a gas discharge, which provides energy to initiate the polymerization of the gaseous monomer and allows a thin polymer film to be deposited on the culture vessel. Cyclic compounds can be used, which may comprise gaseous plasma generated by glow discharge methods. Derivatives of these cyclic compounds, such as 1,2-diaminocyclohexane, are also typically polymerized in gaseous plasma.

[0193] Mixtures of polymerizable monomers can be used. Furthermore, polymerizable monomers can be mixed with other gases that are generally considered non-polymerizable, such as argon, nitrogen, and hydrogen.

[0194] Any culture vessel suitable for adherent culture is considered. Preferred cell culture vessel configurations considered in this disclosure include multiwell plates (e.g., 6-well, 12-well, and 24-well plates), culture dishes (e.g., Pirelli dishes), test tubes, culture flasks, roller flasks, tubes, or shake flasks, etc.

[0195] Materials used for cell culture surfaces may include plastics (e.g., polystyrene, acrylonitrile butadiene styrene, polycarbonate); glass; microporous filters (e.g., cellulose, nylon, glass fiber, polyester, and polycarbonate); materials for bioreactors used in batch or continuous cell culture or genetic engineering (e.g., bioreactors), which may include hollow fiber tubes or microcarrier beads; polytetrafluoroethylene (PTFE). Ceramic and related polymer materials.

[0196] In certain respects, the cell cultures contain little or no extracellular matrix proteins, such as laminin, fibronectin, hylocinin, and matrix. TM Tenosynovin, nestin, platelet-reactive protein, elastin, gelatin, collagen, fibrinogen, partitioning protein, anchoring protein, chondrocyte adhesion protein, connexin, bone sialic acid, osteocalcin, osteopontin, epiinectin, hyaluronic acid, crude fiber regulator, epidermal integrator ligand protein, and hindbrain protein.

[0197] D. Differentiation of HPC into microglia

[0198] Microglia are innate immune cells of the central nervous system, playing a crucial role in brain development, homeostasis, and immune regulation. They are difficult to obtain from human fetuses and neonatal tissues. In some embodiments, the method of the present invention describes the generation, characterization, and cryopreservation of human iPSC-derived microglia (iMGLs) from additionally reprogrammed HPCs under defined conditions. Cryopreserved iMGLs retain purity, secrete immunomodulatory cytokines, and phagocytose pHrodo Red-labeled bacterial bioparticles and amyloid β aggregates. The ability to generate a virtually unlimited number of iMGLs offers tremendous promise for accelerating human neuroscience research into the role of microglia in both normal and disease states.

[0199] In an exemplary method, fresh or frozen HPCs are thawed and plated in a microglia differentiation medium containing FLT-3 ligand and IL-3. Cells can be cultured at a concentration of 10-50 K / cm². 2 For example, 20-35K / cm 2 Density plating. Microglial cell differentiation medium may contain IL-34, TGFβ1, or M-CSF (MDM). Cultures can be grown in MATRIGEL. TM Differentiation can be performed on coated plates or charged surfaces (e.g., Primaria plates, ultra-low adhesion plates, tissue culture plates (TC), or non-tissue culture plates (Non-TC)) and can be high-throughput, such as 96-well plates (e.g., 200 μl microglia differentiation medium per well). During the subsequent 23 days of differentiation, cells can be fed semi-feeded every 48 hours with 50 μl of 2X microglia differentiation medium (MDM) per well. Specifically, differentiation occurs in the absence of ECM proteins (e.g., [missing information]). The procedure was performed under the following conditions. Cells were collected with cold PBS on day 23, and the total viable cell count was quantified using an automated cell counter. Cells were stained to target the surface expression of CD11b, CD11c, CD45, CD33, and TREM-2, and the intracellular expression of TREM-2, IBA, CX3CR1, P2RY12, and TMEM119.

[0200] E. Endothelial cells

[0201] Maintaining MATRIGEL in the presence of E8 TMiPSCs on blebbistatin can adapt to hypoxia for at least 5-10 generations. Cells can be separated from subconfluent iPSCs and seeded onto amine culture dishes at a density of 250,000 cells / well in serum-free (SFD) medium supplemented with 5 μM blebbistatin or 1 μM H1152. 24 hours after seeding, SFD medium supplemented with 50 ng / ml BMP4, VEGF, and FGF2 can be added to the culture. Cells can be fed every 48 hours throughout the differentiation process. The entire process can be carried out under hypoxic conditions. Cells harvested at the end of differentiation can be cryopreserved or incubated at 25 kJ / cm². 2 The density is redeposited on the carboxyl surface to initiate endothelial differentiation in the presence of VascuLife VEGF endothelial medium or SFD endothelial medium.

[0202] In the exemplary method, under VascuLife VEGF endothelial medium or SFD endothelial medium and hypoxic conditions, frozen HPC or live cultures on day 6 are incubated at 25 kJ / cm³. 2 Cells were plated on carboxyl-containing surfaces. Fresh endothelial culture medium was fed to the cells 24 hours after plating, and the cultures were replenished every 48 hours until they reached confluence. Cell confluence may take 5-6 days. Cells were harvested using TrypLE Select, stained for surface endothelial markers CD31, CD105, and CD144, and then plated with endothelial culture medium at 25 kJ / cm². 2 Re-plate the cells onto the carboxyl surface and place them in an anaerobic incubator. On days 2, 4, and 6 after separation, provide the cells with a complete feed of endothelial culture medium. On day 7, harvest the cells, stain them, and re-plate them three more times in the same manner.

[0203] In some embodiments, endothelial cells are transformed into brain microvascular endothelial cells. In an exemplary method, live or cryopreserved HPCs (e.g., day 7 HPCs derived on an amine surface in the presence of SFD supplemented with BMP4, VEGF, and FGF2) are plated onto ECM containing fibronectin (e.g., 50-200 μg / mL, particularly 100 μg / mL) and collagen I (e.g., 100-500 μg / mL, particularly 400 μg / mL) in ECRA medium (human endothelial SFM [Gibco], 1% bovine serum derived from thrombocytopenic plasma [Fisher], 20 ng / mL bFGF [Promega], 10 μM retinoic acid). Cells can be introduced at a concentration of 50-100 kJ / cm². 2 Especially 75k / cm 2The cells are plated at a specific density. Cultures can be maintained under hypoxic incubator conditions. Cultures can be fed ECRA medium every other day until confluence. The confluenced cultures are then harvested, for example, using TrypLE. The harvested cells can be stained to detect PECAM-1 (CD31) and GLUT-1. The harvested cells can be replated, for example, on Transwell inserts with ECRA medium and placed in hypoxic incubator conditions. Cultures can be fed ECRA medium every other day until confluence. Transendothelial resistance (TEER) of the confluenced cultures can be measured.

[0204] F. Mesenchymal cells

[0205] In some implementations, iPSCs differentiate into MSCs. For example, Figure 5C A schematic diagram of the 2D HPC differentiation process for generating MSCs is shown. This will be demonstrated in the presence of E8 in MATRIGEL. TM iPSCs maintained on blebbistatin were adapted to hypoxia for at least 5-10 passages. Cells were separated from subconfluent iPSCs and plated onto amine culture dishes at a density of 250,000 cells / well in serum-free fractionated (SFD) medium supplemented with 5 μM blebbistatin or 10 μM H1152. 24 hours after platening, SFD medium supplemented with 50 ng / ml BMP4, VEGF, and FGF2 was added to the culture. Cells were fed every 48 hours throughout differentiation. The entire process was performed under hypoxic conditions. At the end of day 6 or 7 of differentiation, cells were placed in GMP-MSC medium. Cells were allowed to grow to confluence and harvested at the end of each passage, then inoculated at 50 K / cm². 2 The density was re-coated onto the amine surface of GMP-MSC medium supplemented with 5 uM blebbistatin or 10 uM H1152 to selectively allow the growth and proliferation of MSCs.

[0206] In some cases, cryopreserved day 6 HPC or live cultures at the end of day 6 differentiation were placed on amine-charged surface plates in MSC medium and the presence of 10 μM H1152. Cells were fed fresh MSC medium 24 hours after plating, and the cultures were fed every 48 hours until they reached confluence. Cell confluence took 5–6 days. Cells were collected using TrypLE and stained for the absence of surface MSC markers CD73, CD44, CD105, CD49d, and endothelial markers CD31 and CD144. Cultures were passaged three times under hypoxic conditions and on an amine surface using the above procedure. Cultures were then transferred to normoxic and normal tissue culture plates at P4.

[0207] In some respects, MSCs can further differentiate into pericytes. In an exemplary method, MSCs are seeded into ScienCell pericyte medium (catalog number: 1201) (e.g., on a 6-well Tissue Culture Plastic (TCP) plate at 1-20 kJ / cm²). 2 Cell density, especially at 10 kJ / cm³ 2 Cells were cultured at a controlled density and placed in a normoxic incubator. Cultures were fed ScienCell periodic cell culture medium every other day until confluence. Confluenced cultures could be harvested, for example, using TrypLE. Harvested cells could be stained to detect glial antigen 2 / chondroitin sulfate proteoglycan (NG2) and PDGFR-β (CD140b). Harvested cells could be replated in ScienCell periodic cell culture medium (e.g., in TCP 6-well plates at 1-20 kJ / cm²). 2 Cell density, especially at 10 kJ / cm³ 2 (Cell density). Cultures can be fed ScienCell weekly cell culture medium every other day until confluence, then harvested and stained in the same manner as described above. Cells are then replated until the culture expands and maintains purity. Furthermore, cells can be positively stained for the presence of CD146, CD49a, CD166, CD54, CD73, CD105, CD13, CD56, CD49d, and / or CD44.

[0208] G. Differentiation medium

[0209] Cells can be cultured using the nutrients necessary to support the growth of each specific cell population. Typically, cells are cultured in a growth medium containing a carbon source, a nitrogen source, and a buffer to maintain pH. The medium may also contain fatty acids or lipids, amino acids (e.g., non-essential amino acids), vitamins, growth factors, cytokines, antioxidants, pyruvate, buffers, pH indicators, and inorganic salts. Exemplary growth media contain essential media such as Duchenne Modified Eagle Medium (DMEM) or Essential 8. TM (E8 TM Essentially essential media are culture media supplemented with various nutrients, such as non-essential amino acids and vitamins, to promote stem cell growth. Examples of essential media include, but are not limited to, the essential essential media Eagle (MEM) α medium, Duchenne modified Eagle (DMEM) medium, RPMI-1640 medium, 199 medium, and F12 medium. Furthermore, essential media may be supplemented with additives, such as equine, calf, or fetal bovine serum. Alternatively, the medium may be serum-free. In other cases, growth media may contain “knockout serum substitutes,” which are referred to herein as serum-free formulations optimized for the growth and maintenance of undifferentiated cells, such as stem cells, in cultures.TM Serum substitutes are disclosed, for example, in U.S. Patent Application No. 2002 / 0076747, which is incorporated herein by reference. Preferably, PSCs are cultured in a fully defined culture medium without a feeder layer.

[0210] In some embodiments, the culture medium may contain or not contain any alternative to serum. Serum alternatives may include materials appropriately containing albumin (e.g., lipid-rich albumin, albumin alternatives such as recombinant albumin, plant starch, dextran, and protein hydrolysates), transferrin (or other iron transporters), fatty acids, insulin, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thioglycerol, or equivalents thereof. For example, serum alternatives can be prepared by the method disclosed in International Publication No. WO98 / 30679. Alternatively, for convenience, any commercially available material may be used. Commercially available materials include KNOCKOUT. TM Serum substitutes (KSR), chemically defined lipid concentrates (Gibco), and GLUTAMAX TM (Gibco).

[0211] Other culture conditions may be appropriately defined. For example, the culture temperature may be about 30 to 40°C, such as at least or about 31, 32, 33, 34, 35, 36, 37, 38, or 39°C, but is not particularly limited thereto. In one embodiment, the cells are cultured at 37°C. The CO2 concentration may be about 1 to 10%, such as about 2 to 5%, or any range therefrom. The oxygen tension may be at least, up to, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 20%, or any range therefrom.

[0212] H. Cryopreservation

[0213] Cells produced by the methods disclosed herein can be cryopreserved at any stage of the process, such as Stage I, Stage II, or Stage III, see, for example, PCT Publication No. 2012 / 149484A2, which is incorporated herein by reference. Cells can be cryopreserved with or without a substrate. In several embodiments, the storage temperature range is about -50°C to about -60°C, about -60°C to about -70°C, about -70°C to about -80°C, about -80°C to about -90°C, about -90°C to about -100°C, and the overlap thereof. In some embodiments, lower temperatures are used for the storage (e.g., maintenance) of the cryopreserved cells. In several embodiments, liquid nitrogen (or other similar liquid coolant) is used to store the cells. In a further embodiment, cell storage is greater than about 6 hours. In another embodiment, cell storage is about 72 hours. In several embodiments, cell storage is about 48 hours to about one week. In other embodiments, cell storage is about 1, 2, 3, 4, 5, 6, 7, or 8 weeks. In a further embodiment, cells are stored for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. Cells may also be stored for longer periods. Cells may be cryopreserved individually or on a substrate (such as any substrate disclosed herein).

[0214] In some embodiments, additional cryoprotectants may be used. For example, cells may be cryopreserved in a cryopreservation solution containing one or more cryoprotectants such as DM80, serum albumin (e.g., human or bovine serum albumin). In some embodiments, the solution contains about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% DMSO. In other embodiments, the solution contains about 1% to about 3%, about 2% to about 4%, about 3% to about 5%, about 4% to about 6%, about 5% to about 7%, about 6% to about 8%, about 7% to about 9%, or about 8% to about 10% dimethyl sulfoxide (DMSO) or albumin. In one specific embodiment, the solution contains 2.5% DMSO. In another specific embodiment, the solution contains 10% DMSO.

[0215] Cells can be cooled during cryopreservation, for example, at a rate of about 1 °C / min. In some embodiments, the cryopreservation temperature is about -80 °C to about -180 °C, or about -125 °C to about -140 °C. In some embodiments, the cells are cooled to 4 °C before being cooled at a rate of about 1 °C / min. Cryopreserved cells can be transferred to the gas phase of liquid nitrogen before thawing for use. In some embodiments, for example, once the cells have reached about -80 °C, they are transferred to a liquid nitrogen storage area. Cryopreservation can also be accomplished using a rate-controlled cryostat. Cryopreserved cells can be thawed, for example, at a temperature of about 25 °C to about 40 °C, and typically at a temperature of about 37 °C.

[0216] III. How to Use

[0217] This disclosure provides a method for generating large numbers of multi-lineage cells. These cell populations can be used for many important research, development, and commercial purposes. These include, but are not limited to, in vivo transplantation or implantation of cells; in vitro screening of antiviral drugs, cytotoxic compounds, carcinogens, mutagens, growth / regulatory factors, and pharmaceutical compounds; elucidating the mechanisms of liver diseases and infections; studying the mechanisms of action of drugs and / or growth factors; diagnosing and monitoring cancer in patients; gene therapy; and the production of bioactive products, to name just a few.

[0218] A. Pharmaceutical Composition

[0219] This document also provides pharmaceutical compositions and formulations comprising the cells and pharmaceutically acceptable carriers of the present invention.

[0220] The cellular compositions according to the invention for administration to subjects can therefore be formulated in any conventional manner using one or more physiologically acceptable carriers, said carriers comprising excipients and adjuvants that facilitate the processing of the compound into a pharmaceutically acceptable formulation. A suitable formulation depends on the chosen route of administration.

[0221] The pharmaceutical compositions and formulations described herein can be made by combining an active ingredient (e.g., cells) with one or more optional pharmaceutically acceptable carriers (Remington's Pharmaceutical Sciences 22). nd(edition, 2012) These are prepared as lyophilized formulations or aqueous solutions. Pharmaceutically acceptable carriers are generally non-toxic to recipients at the doses and concentrations used, and include, but are not limited to: buffers such as phosphates, citrates, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzyl chloride; phenol, butanol, or benzyl alcohol; alkyl esters of p-hydroxybenzoate such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues). Polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents, such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; counterions that form salts, such as sodium; metal complexes (e.g., zinc-protein complexes); and / or nonionic surfactants, such as polyethylene glycol (PEG). Exemplary pharmaceutically acceptable carriers further include interstitial drug dispersants, such as soluble neutral active hyaluronidase glycoprotein (sHASEGP), such as human soluble PH-20 hyaluronidase glycoprotein, such as rHuPH20 (…). Baxter International, Inc. Certain exemplary sHASEGPs and methods of use, including rHuPH20, are described in U.S. Patent Publications 2005 / 0260186 and 2006 / 0104968. In one aspect, sHASEGP is combined with one or more additional glycosaminoglycans, such as chondroitinase.

[0222] B. Distribution for commercial, therapeutic, and research purposes

[0223] In some embodiments, a reagent system is provided that includes cells present at any time during manufacturing, distribution, or use. The kit may contain any combination of the cells described in this disclosure with undifferentiated pluripotent stem cells or other differentiated cell types (generally sharing the same genome). Each cell type may be packaged together, in the same facility or at different locations, at the same or different times, or in separate containers, under the control of the same entity or different entities sharing a business relationship. The pharmaceutical composition may optionally be packaged in a suitable container with written instructions for the desired purpose (e.g., mechanistic toxicology).

[0224] In some embodiments, a kit is provided that may include, for example, one or more culture media and components for generating cells. Where suitable, the reagent system may be packaged in an aqueous medium or lyophilized form. The container device of the kit typically includes at least one vial, test tube, flask, bottle, syringe, or other container device into which the components are placed and preferably appropriately aliquoted. When the kit contains more than one component, it will typically also include a second, third, or other additional container in which the additional components may be placed separately. However, a combination of various components may be contained in the vial. The components of the kit may be provided as a dry powder. When reagents and / or components are provided in dry powder form, the powder can be reconstituted by adding a suitable solvent. It is also considered that the solvent may be provided in another container device. The kits of this disclosure will also typically include means for containing the kit components in a closed space for commercial sale. Such a container may include an injection or blow-molded plastic container in which the desired vial is held. The kit may also include instructions for use, such as in printed or electronic formats, such as digital formats. Example

[0225] The following embodiments illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the following embodiments represent techniques discovered by the inventors that work well in the practice of the invention, and therefore can be considered to constitute preferred modes of practice. However, those skilled in the art, based on this disclosure, will understand that many changes can be made to the specific embodiments disclosed without departing from the spirit and scope of the invention, and similar or analogous results can still be obtained.

[0226] Example 1 – Production of Endothelial Cells

[0227] To maintain MATRIGEL in the presence of E8 TM iPSCs on blebbistatin or blebbistatin were adapted to hypoxia for at least 5-10 passages. Cells were separated from subconfluent iPSCs and plated onto amine culture dishes at a density of 250,000 cells / well in the presence of serum-free fractional-derived (SFD) medium supplemented with 5 μM blebbistatin or 1 μM MH1152. 24 hours after plating, SFD medium supplemented with 50 ng / ml BMP4, VEGF, and FGF2 was added to the culture. Cells were fed every 48 hours throughout differentiation. The entire process was performed under hypoxic conditions. Cells were harvested at the end of differentiation and could be cryopreserved or inoculated at 25 kJ / cm² in the presence of VascuLife VEGF endothelial medium or SFD endothelial medium. 2 The density is redeposited on the carboxyl surface to initiate endothelial differentiation. Figure 2 ).

[0228] In the presence of 1 μM H1152 and hypoxia, and in the presence of VascuLife VEGF endothelial medium, frozen HPC or live cultures on day 6 were incubated at 25 kJ / cm². 2 Cells were plated on carboxyl-containing surfaces. Fresh VascuLife feed was provided to the cells 24 hours after plating, and the cultures were replenished every 48 hours until confluence was achieved. Cell confluence takes 5–6 days. Cells were harvested using Accumax with minimal stirring or pipetting, stained for the surface endothelial markers CD31, CD105, and CD144, and treated with VascuLife + H1152 at 25 kJ / cm². 2 Cells were replated onto a carboxyl surface and placed in an anaerobic incubator. On days 2, 4, and 6 post-isolation, cells were fed a complete VascuLife feed. On day 7, cells were harvested, stained, and replated three more times in the same manner. Histograms depict the increase in endothelial cell purity at each replate stage. Pure endothelial cells were produced using continuous passage purification without the use of CD31+MACS. Endothelial cells could be cryopreserved at the end of the third replate passage. Figure 3 ).

[0229] Example 2 – Generation of Mesenchymal Stem Cells

[0230] Figure 5C A schematic diagram of the 2D HPC differentiation process for MSC generation is shown. This is achieved by maintaining the MATRIGEL in the presence of E8. TM iPSCs on blebbistatin or hydrin were adapted to hypoxia for at least 5-10 passages. Cells were separated from subconfluent iPSCs and plated onto amine culture dishes at a density of 250,000 cells / well in serum-free fractional-determined (SFD) medium supplemented with 5 μM blebbistatin or 1 μM H1152. 24 hours after plating, SFD medium supplemented with 50 ng / ml BMP4, VEGF, and FGF2 was added to the culture. Cells were fed every 48 hours throughout differentiation. The entire process was performed under hypoxic conditions. At the end of day 6 / 7 of differentiation, cells were transferred to GMP-MSC medium. Phenotypic analysis of the precursor population was performed post-harvest. Figure 5C Allow cells to grow to confluence and harvest at the end of each passage, then incubate at 50 K / cm². 2 The density was re-coated onto the amine surface of GMP-MSC medium supplemented with 5 uM blebbistatin or 1 uM H1152 to selectively allow the growth and proliferation of MSCs.

[0231] Live cultures from cryopreserved day 6 HPCs differentiated from 3D / 2D HPCs, or from day 6 of 2D HPCs at the end of differentiation, were placed on amine-charged surface plates in MSC medium and 1 μM H1152. Cells were fed fresh MSC medium 24 hours after plating, and cultures were fed every 48 hours until confluence. Cell confluence took 5–6 days. Cells were collected using TrypLE and stained for the absence of surface MSC markers CD73, CD44, CD105, CD49d, and endothelial markers CD31 and CD144. Cultures were passaged three times under hypoxic conditions and on an amine surface using the above procedure. Cultures were transferred to normoxic and normal tissue culture plates at P4. MSC purity specifications were achieved at P6 (…). Figure 6 , 7 Thaw the MSCs frozen at P3 and place them in a container as follows: Figure 8A The lineage-specific differentiation matrix described herein was used to demonstrate the trilineage potential for producing osteocytes, chondrocytes, and adipocytes. Figure 8B (By using 1000 cells / cm in a 10cm tissue culture plate). 2 The density of MSCs plated in slabs demonstrated the clonal proliferation capacity of cryopreserved MSCs. Cells were fed with MSC medium for two weeks, with the medium changed every other day. Colonies were stained with crystal violet and scored. Figure 8C ).

[0232] Example 3 – Generation of pericytes from MSCs

[0233] iCell MSC and iPSC-derived pericytes were sampled at 50% confluence and analyzed by flow cytometry for known pericyte markers PDGFRβ, NG2, and CD146. Cryopreserved MSCs were thawed and analyzed at 35,000 cells / cm³. 2 In the absence of extracellular matrix (ECM), MSCs were plated in 6-well plates in maintenance medium. Figure 9A The cells were brought together and dispersed at a rate of 15,000 cells / cm². 2 Re-plate in 6-well plates in SFD pericyte culture medium (SPM) without extracellular matrix (ECM). Figure 9A ; Figure 9B ).

[0234] Primary human cerebral perivascular cells (HBVP) (ScienCell#1200) were thawed and stored at 5,000 cells / cm². 2Periperocytes were plated in ScienCell #1201 medium on poly-L-ornithine-coated 6-well plates. These cells served as positive controls during differentiation. Periperocytes derived from ScienCell HBVP, iCell MSCs, and iPSCs were analyzed by flow cytometry for known periperocyte markers PDGFRβ, NG2, and CD146. Figure 9C Pericylic acid markers of iCell MSCs were absent upon thawing. HBVP and iPSC-derived pericytes showed expression of known pericytic markers PDGFRβ, NG2, and CD146, with iPSC-derived pericytes exhibiting higher purity than ScienCell HBVP. Figure 9C iPSC-derived pericytes exhibited a morphology similar to ScienCell HBVP. Figure 9D ).

[0235] Based on their function, pericytes can be classified into phenotypes PC1 (pro-inflammatory) or PC2 (contractile) (Rustenhoven et al., 2017). The two subtypes are characterized by... Figure 9E As described in the text. After thawing, iPSC-derived pericytes were subtyped by flow cytometry for PC1 and PC2 markers CD274, VCAM1, calmodulin, desmin, DLK1, and αSMA. Figure 9F iPSC-derived pericytes revealed characteristics of contractile pericytes (PC2 subtype).

[0236] In addition to the nonspecific phagocytic uptake observed in chronic and acute BBB models, pericytes also specifically modulate their neuronal microenvironment by processing the clearance of certain macromolecules under physiological and pathological conditions (Winkler et al., 2014). iPSC-derived pericytes were cultured at 15,000 cells / cm². 2 Cells were plated in 96-well plates with a PDL coating (Greiner #655946) using SPM. Cells were allowed to rest for three days after plating before the addition of the death indicator NucGreen Dead 488 (Invitrogen #R37109) and Staphylococcus aureus pHrodo Red bioparticles (Invitrogen #A10010). The plates were placed on an IncuCyte real-time imaging system for over a month, with weekly replenishment (including the same concentrations of live / dead and bioparticle reagents). iPSC-derived pericytes showed observable phagocytic activity higher than the control Staphylococcus aureus bioparticles.

[0237] Example 4 - Generation of Brain Microvascular Endothelial Cells (BMEC)

[0238] To maintain MATRIGEL in the presence of E8 TM iPSCs on fibronectin are adapted to hypoxia for at least 5-10 generations to generate brain microvascular endothelial cells. Live or cryopreserved HPCs (e.g., day 6 HPCs derived on an amine surface in the presence of SFD supplemented with BMP4, VEGF, and / or FGF2, e.g., BMP4 and FGF2) are plated onto ECM containing fibronectin (e.g., 50-200 μg / mL, especially 100 μg / mL) and collagen IV (e.g., 100-500 μg / mL, especially 400 μg / mL) in ECRA medium (human endothelial SFM (Gibco), 1% platelet-deficient plasma-derived bovine serum (Fisher), 20 ng / mL bFGF (Promega), 10 μM retinoic acid). Cells are grown at 50-100 kJ / cm². 2 Especially 75k / cm 2 The cells were plated at a specific density. Cultures were fed daily and maintained in an anoxic incubator. Cultures were fed ECRA medium every other day until confluence. The confluenced cultures were then harvested, for example, using TrypLE. The harvested cells were stained to detect PECAM-1 (CD31) and GLUT-1 to confirm the identity of BMECs. Figure 10B The harvested cells were replated, for example, on Transwell inserts containing ECRA medium, and placed in an anaerobic incubator. Figure 10A The culture can be fed ECRA medium every other day until confluence. It can be analyzed by flow cytometry. Figure 10C ) and immunocytochemistry ( Figure 10D The presence of P-gp, CD105, Glu-1, and CD31 expression in confluent cultures was measured using transendothelial resistance (TEER) assays and compared with blank culture medium. Figure 10E For immunohistochemistry, cells were washed three times with 200 μl DPBS and then incubated overnight at 4°C with rabbit anti-P-gp antibody (1:50 in blocking buffer (10% FBS, 0.01% Triton X in DPBS)). After washing three times with 200 μl DPBS, P-gp was stained with secondary antibody (1:1000, donkey anti-rabbit IgG Alexa Fluor 488 (Invitrogen)). Cell nuclei were stained with Hoechst 3342 (Thermo Fisher). Images were captured at 200x magnification using ImageXpress (Molecular Devices, LLC).

[0239] Example 5 - Production of microglia

[0240] To maintain MATRIGEL in the presence of E8 TM Alternatively, iPSCs on blebbistatin can adapt to hypoxia for at least 5-10 generations. 2DHPC differentiation: Cells are separated from sub-confluent iPSCs and seeded onto amine culture dishes at a density of 250,000-500,000 cells / well in serum-free fractional-derived (SFD) medium supplemented with 5 μM blebbistatin or 1 μM H1152. 24 hours after seeding, SFD medium supplemented with 50 ng / ml BMP4, VEGF, and FGF2 is added to the culture. A complete medium exchange is performed the following day.

[0241] On day 5 of differentiation, cells were placed in a culture medium containing 50 ng / ml Flt-3 ligand, SCF, TPO, IL3 and IL6, and 5 U / ml heparin. Cells were fed every 48 hours throughout the differentiation process. The entire process was conducted under hypoxic conditions. HPC was quantified by the presence of CD43 / CD34 cells.

[0242] 3D HPC Differentiation: Cells were separated from sub-confluent iPSCs and seeded into spin flasks at a density of 250,000-500,000 cells / ml in serum-free component-determined (SFD) medium supplemented with 5 μM blebbistatin or 1 μM MH1152. The SFD medium was replaced with 50 ng / ml BMP4, VEGF, and FGF2 supplemented 24 hours after seeding. On day 5 of differentiation, cells were placed in medium containing 50 ng / ml Flt-3 ligand, SCF, TPO, IL3, and IL6, and 5 U / ml heparin. Cells were fed every 48 hours throughout the differentiation process. The entire process was performed under hypoxic conditions. HPC was quantified by the presence of CD43 / CD34. Process overview and efficiency are shown in (…). Figure 11 Examples are given in () and the composition of the culture medium is described in () Figure 12 Listed in ).

[0243] HPC was placed in microglial cell differentiation medium MDM or 2X-MDM. Figure 11 Cultures were fed every 48 hours. Purity markers of microglia cultures on day 23 of differentiation were quantified before and after cryopreservation. Figure 13A , Figure 13B ).

[0244] The purity of live and cryopreserved microglia cultures at day 23 was assessed. Microglia cultures at differentiation were harvested at day 23 and stained for the presence of microglia-specific markers. The remaining cells were cryopreserved using a rate-controlled cryostat. The cryopreserved cells were thawed and stained for the presence of microglia-specific markers. For both groups, cell surface expression of CD45, CD33, TREM2, and CD11b was assessed. Figure 14 The intracellular expression of A) and PU.1, IBA, P2RY12, TREM2 and TMEM119 was determined by flow cytometry. Figure 14 B). The results showed that cryopreserved microglia maintained their purity after cryopreservation.

[0245] HPCs were placed in culture medium in the presence of MDM to initiate microglia differentiation, and intermittent feeding with 2X-MDM was performed. Cells were cryopreserved on days 20, 23, and 26 of differentiation using either a manual freezing protocol or a rate-controlled cryostat (CRF). Cryopreserved cells were transferred to liquid nitrogen for one week. Cryopreserved microglia were thawed and placed in microglia maturation medium (MMM). Cultures were fed with fresh microglia maturation medium every 48 hours. Cells were harvested on days 3, 5, 7, 10, 12, and 14 post-thawing, and viable cell recovery relative to the initial plate number was quantified. Figures 15A-15C ).

[0246] In the presence of MDM, cryopreserved HPCs were differentiated into microglia. The total number of viable HPCs inputted and output microglia was quantitatively measured. Process efficiency was calculated by dividing the purity and absolute number of TREM2-positive cells present on day 23 of microglia differentiation by the absolute number of input viable HPCs. Figure 16 ).

[0247] Microglia cryopreserved on day 20 (Fig. 17A), day 23 (Fig. 17B), or day 26 (Fig. 17C) of differentiation were thawed in microglia maturation medium (MMM) and fed fresh medium every 48 hours. Total viability and absolute cell count were quantified on days 3, 7, and 10 post-thawing. Data showed that microglia on day 23 had a higher recovery rate after thawing than microglia on day 26. Figures 17A-17C ).

[0248] Next, the cryopreserved microglia were functionally assessed on days 20, 23, and 26 of the differentiation process. Cells were thawed and seeded at 15,000 viable cells / well in 96-well plates with 200 μl of microglia maturation medium per well. Cells were treated with diluted 1 μg / well of either conditioned or unconditioned pHrodo Red bioparticles (ThermoFisher #A10010, 2 mg per vial; stored at -20°C). The plates were placed on IncuCyte, and images of phagocytosis were captured at different time points up to 5 days after thawing. Cells cryopreserved using a rate-controlled freezing method exhibited stronger phagocytosis (due to higher cell viability). Figure 19 )).

[0249] Functional assessment was extended to later time points post-thawing. Phagocytic potential was assessed at days 5, 7, and 14 post-thawing using real-time imaging on the IncuCyte system for microglia cryopreserved at days 20, 23, and 26 of differentiation, either manually or with a rate-controlled cryostat. Cryopreserved microglia were thawed and plated in MMM for three days. Viable cell counts at the end of three days were determined as shown in Figure 18B. 15,000 viable cells were plated in 96-well plates with 200 μl of microglia maturation medium (MMM) per well and diluted 1 μg / well of conditioning or unconditioned pHrodo Red bioparticles. The plates were then placed on IncuCyte, and images of phagocytosis were obtained at various time points up to 5, 7, and 14 post-thawing. The manual cryopreservation method showed the rate of decrease / right shift in phagocytosis (due to reduced cell viability) under all conditions.

[0250] The phagocytic index is a measure of phagocytic activity, determined by calculating the number of bacteria taken up by each phagocyte during a limited incubation period of a suspension of bacteria and phagocytes. The ability of cryopreserved microglia to phagocytose labeled bacterial particles is quantified by the ratio of the number of phagocytosed red objects to the total number of viable cells. This ratio is defined as the phagocytic index (PQI). Figure 21 ).

[0251] Cryopreserved microglia were thawed and seeded at 15,000 viable cells / well in 96-well plates with 200 μl of microglia maturation medium per well. Cells were treated with diluted 1 μg / well of either conditioned or unconditioned pHrodo Red bioparticles (Thermo Fisher #A10010, 2 mg per vial; stored at -20°C). Plates were placed on IncuCyte, and images of phagocytosis were captured at different time points up to 5 days after thawing. Cells cryopreserved using a rate-controlled freezing method exhibited stronger phagocytosis (due to higher cell viability). Figure 22 )).

[0252] Next, HPC differentiation into microglia was further developed in the absence of ECM and in a 96-well format suitable for screening applications. Differentiation was performed in ultra-low attachment (ULA), tissue culture (TC), and non-tissue culture (Non-TC) containers (Fig. 23A). Cryopreserved HPCs were cultured at 20,000–35,000 viable cells / cm² in the presence of 200 μL microglia differentiation medium per well. 2 The cells were densely seeded in 96-well Primaria plates or ultra-low attachment, tissue culture (TC) or non-tissue culture (Non-TC) plates per well (Fig. 23B). During the next 23 days of differentiation, 50 μl of medium was added to each well of MDM every 48 hours. Cells were collected with cold PBS on day 23, and the total viable cell count was quantified using an automated cell counter. Cells were stained for surface expression of CD11b, CD45, CD33, and TREM2, and for intracellular expression of TREM2, IBA, P2RY12, and TMEM119. Figures 24A-24B ).

[0253] Table 1: Process efficiency for generating microglia on charged surfaces.

[0254] Board type Day 0 cell count Cell count on day 23 Amplification Primera 0.684×106 4.0×106 5.8x ULA 0.684×106 2.06×106 3x TC 0.684×106 3.22×106 4.7x Non-TC 0.684×106 3.52×106 5.1x

[0255] Cytokines and chemokines released from cryopreserved microglia. Microglia cryopreserved on day 23 were thawed into MDM medium and seeded at 50,000 cells / well in Primaria 96-well plates. Cells were seeded for three days prior to stimulation with 100 ng / ml LPS and 50 ng / ml interferon-gamma. Stimulation was performed in triplicate and lasted 24 hours. The supernatant was centrifuged to remove cells and debris and immediately incubated at -20°C. The supernatant was analyzed using a multiplex Luminex assay.

[0256] Example 6 - Engineering iPSCs to generate variants mimicking neurodegenerative diseases

[0257] TREM2 function was disrupted by introducing an insertion / deletion in exon 2, leading to frameshift and premature termination of translation. The TAL nuclease (paired with TREM2 below) was engineered to bind to a DNA sequence centered at amino acid 58 within exon 2. The cell line used for engineering was FCDI iPSC line 01279.107. The TAL nuclease mRNA and a co-selection plasmid expressing blast fungicide resistance under the control of the SV40 promoter were electroporated into cells using a BioRad Gene PulserXcell system set to 125V / 950uF. Cells were plated and short blast fungicide selections were applied on days 1 and 2 post-electroporation. Surviving cells were grown and then single cells were sorted into 96-well plates on day 7 post-electroporation. After approximately two weeks, 81 clones were selected and genotyped by PCR and sequencing.

[0258] Of the 81 clones sequenced, 7 showed sequence modifications. Three clones contained one allele with a 1-base-pair insertion, three clones contained one allele with a 1-base-pair deletion, and one clone was a compound heterozygote with one allele containing a 1-base-pair insertion and one allele containing a 4-base-pair deletion. The seventh clone contained a 24-base-pair deletion that was not expected to introduce a frameshift. The clones were amplified, cryopreserved, and subjected to sequence confirmation and karyotype analysis. After differentiation into microglia, two major clones were selected as examples of heterozygous or homozygous disruption. The heterozygous clone 01279.1185 contained an allele with a 1-bp insertion, resulting in a frameshift at position 60 of TREM2 and termination after 45 subsequent amino acids. Homozygous clone 01279.1187 contains an allele with a 1 bp frameshift insertion at position 59 that terminates after 16 amino acids, and a second allele with a 4 bp deletion at position 59 that results in a frameshift and terminates after 46 amino acids.

[0259] Table 2: Target sequences.

[0260] TAL target sequence TREM2-F GGTGCCGCCAGCTGGGAG TREM2-R GCGTGCTGACCACACGCT

[0261] Table 3

[0262]

[0263] Example 7 - Generation of additional genetically engineered lines simulating neurodegeneration:

[0264] The Parkinson's disease model was generated through nuclease-mediated homologous recombination and the genetically engineered iPSC 01279, derived from the donor oligonucleotide SJD 14-133 via weight-programming. The resulting iPSC contained SNP rs104893877, in which amino acid 53 was changed from alanine to threonine, resulting in the A53T variant in the α-synuclein gene (SNCA), and two silencing mutations leading to the SNCA A53TiPSC lineage.

[0265] An isogenetic model for studying Rett syndrome was generated using nuclease-mediated homologous recombination and donor plasmid p1553. Donor plasmid p1553 inserts a series of stop codons before the methyl CpG-binding domain, followed by a PGKp-PuromycinR-SV40pA selector cassette with a LoxP site lateralized. The MECP2HM line, derived from parental line 01279, provides a disease model for Rett syndrome.

[0266] HPCs and microglia were generated from isogenetic engineered iPSCs: homozygous and heterozygous TREM2KO iPSCs derived from 01279 iPSCs, as well as SNCA A53T and MECP2 HM engineered lines derived from 01279, in E8 and MATRIGEL. TM Cells were maintained in the presence of [unspecified substance] and acclimated to hypoxic conditions by passage 10 times. Karyotype analysis was performed, and HPC differentiation was initiated from the iPSC library using a 3DHPC differentiation protocol. Cells were separated from subconfluent iPSCs and seeded into spin flasks at a density of 250,000–500,000 cells / mL in serum-free component-determined (SFD) medium supplemented with 5 μM blebbistatin or 1 μM H1152. The SFD medium was replaced with 50 ng / mL BMP4, VEGF, and FGF2 24 hours after seeding. On day 5 of differentiation, cells were placed in medium containing 50 ng / mL Flt-3 ligand, SCF, TPO, IL3, and IL6, and 5–10 U / mL heparin. Cells were fed every 48 hours throughout the 13-day differentiation period. The entire process was performed under hypoxic conditions. HPC was quantified by the presence of CD43 / CD34. HPC was cryopreserved after MACs sorting using CD34 beads. Microglia were generated by thawing cryopreserved HPC and placing the cells in a 23-day differentiation process as described in Example 5.

[0267] Cryopreserved microglia from day 23 wild-type and TREM-engineered clones were thawed, and TREM-2 expression, along with the presence of CD45, was quantified by flow cytometry. Figure 26 ).

[0268] Microglia derived from genetically engineered lines were thawed on day 23 of cryopreservation and stained for the presence of microglia-specific markers. Cells were stained by flow cytometry to quantify the cell surface expression of CD45, CD33, TREM2, and CD11b, as well as the intracellular expression of PU.1, IBA, P2RY12, TREM2, and TMEM119 proteins. Figure 26 The purity levels obtained from all four types of isogenetic-engineered iPSCs were summarized. The results show that high-purity microglia can be generated from isogenetic-engineered iPSCs without changing the differentiation protocol.

[0269] The levels of soluble TREM2 (sTREM2) protein secreted by thawed microglia were quantified from conditioned medium collected from WT and TREM2 heterozygous and homozygous KO mutants using Simple Step ELISA (AbCam). Figure 27A WT and TREM2 KO microglia were thawed and plated at the same density in mature medium in 96-well Primaria plates. Used medium was collected on days 3 and 7 post-thaw. Cultures were fed semi-feeded with fresh mature medium on days 3 and 5 post-thaw. Data revealed differences in soluble TREM2 levels among WT, heterozygous TREM2 KO, and homozygous KO microglia. This assay can be used as a functional assay to differentiate between WT and TREM2 engineered iPSCs.

[0270] The levels of soluble TREM2 (sTREM2) protein secreted by thawed microglia were quantified using Simple Step ELISA (AbCam) from conditioned medium collected from WT and TREM2 heterozygous and homozygous KO mutants, MECP2HM, and SNCA-A53T. Figure 27A WT and TREM2KO microglia were thawed and seeded at the same density in mature medium in 96-well Primaria plates. Used medium was collected on days 3 and 7 post-thaw. Cultures were fed half-feed with fresh mature medium on days 3 and 5 post-thaw. Data revealed differences in soluble TREM2 levels among WT, heterozygous TREM2 KO, and homozygous KO microglia. This assay can be used as a functional assay to distinguish between WT and TREM2 engineered iPSCs. Release of soluble TREM2 was impaired in A53T-SNCA microglia, while MECP2HM microglia did reveal any alteration in the level of sTREM released in the medium. Figure 27B ).

[0271] Cytokines and chemokines released from cryopreserved microglia by genetically engineered microglia. Microglia cryopreserved on day 23 from WT, 1185HT TREM2 KO, 1187HO TREM2 KO A53T-SNCA, and MeCP2HM microglia were thawed into MDM medium and seeded at 50,000 cells / well in Primaria 96-well plates. Cells were seeded for three days prior to stimulation with 100 ng / ml LPS to examine M1-mediated responses. Stimulation was performed in triplicate and lasted 24 hours. The supernatant was centrifuged to remove cells and debris and immediately placed at -20°C. The supernatant was analyzed using a multiplex Luminex assay. The results of this multiplex Luminex assay are captured as a heatmap in Figure 27C. The engineered lines secreted higher levels of IL-6 compared to the ANH control. Compared to ANH, TREM2 HZ, TREM2 HO, and MeCP2HM microglia released less TNFα but increased IL-6 levels. A53T-SNCA microglia released similar levels of IL-6 and TNFα compared to AHN control microglia.

[0272] When treated with M1 stimulant (LPS), all engineered lines released the M2 cytokine IL-10. MECP2HM microglia released less IL-10 compared to AHN control microglia (Fig. 27E). Both AHN and engineered microglia responded to LPS stimulation by releasing CCL2 / MCP-1, CCL20 / MIP-3α, CCL4 / MIP-1β, CCL5 / RANTES, CX3CL1 / fractal chemokine, CXCL1 / GROα, CXCL10 / IP-10, CXCL2 / GROβ, and IL-8 / CXCL8. Some inherent differences existed in cytokine release levels. TREM2HO revealed the highest levels of CCL4, a key analyte released during Alzheimer's disease (AD) flare-ups. MECP2HM, TREM2HZ, and TREM2HO microglia released higher levels of CXCL1 / GRO, indicating an attempt to recruit helper cell type granulocytes to aid in killing microbes and triggering an inflammatory response during phagocytosis. MECPHM microglia showed spontaneous secretion of IL-8 / CXC18. This analyte is elevated in brain injury and induces the expression of pro-inflammatory proteases and MMP-2 and MMP-9. MECPHM microglia secreted higher levels of IL-6. These results suggest that MECPHM is primed for a pro-inflammatory response. Engineered and AHN microglia released similar levels of PDL-1, CD40, FLT-3, and PDGFAA in response to LPS in culture medium.

[0273] Prior to screening experiments, cryopreserved microglia were thawed in maturation medium and allowed to recover for 48 hours (Fig. 36). 5,000 microglia from TREM2 WT and TREM2 HOKO were seeded in 40 μL of medium in each well of a 384-well plate for 24 hours. In the first group (plate 1), cells were pretreated with a compound at a final concentration of 1 μM. 24 hours after compound treatment, pHrodo-labeled amyloid-β was added to plate 1 at a final concentration of 1 μM, and the cells were captured and phagocytosed on IncuCyteS3 plates for 96 hours (Figs. 37–39). In the second group (plate 2), cells were seeded for 24 hours and then treated with 1 μg / mL LPS at a final concentration of 1 μg / mL (Figs. 40–42). 24 hours after LPS exposure and 48 hours after initial seeding, pHrodo-labeled amyloid-β was added to plate 2 at a final concentration of 1 μM. Cells were imaged hourly using IncuCyte for up to 96 hours. Phagocytic data were captured as total red object intensity × μM² / image. The final volume remained constant across all treatments. The results of the screening are summarized in... Figure 43 middle.

[0274] To understand the cytokines required for microglia survival after thawing in mature culture media, a schematic matrix of 32 different culture medium formulations was designed. Figure 28 WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia were placed at a density of 15,000 viable cells in 250 μl of microglia basal medium or MMM in 96-well plates, or microglia basal medium supplemented with a single cytokine (Fig. 29), two cytokines (Fig. 30), three cytokines (Fig. 31), or four cytokines (Fig. 32) in mature medium. Cell viability kinetics were captured on the IncuCyte system. NucGreen Dead diluted to 2 drops / mL was added to all wells containing cells with various medium components to capture the number of dead cells over time. Images were captured every 8 hours for 72 hours without any intermittent feeding. The intensity of NucGreen Dead quantified the number of dead cells in the culture.

[0275] WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia were placed at a density of 15,000 viable cells in 250 μl of basal microglia culture medium (Fig. 33A) or MMM (Fig. 33B), IL-34-supplemented basal microglia culture medium (Fig. 36C), IL-34-supplemented basal microglia culture medium (Fig. 33D), MCSF-supplemented basal microglia culture medium (Fig. 33D), or IL-34-supplemented basal medium (Fig. 33C), IL-34-supplemented basal medium, or a combination of IL-34 and MCSF (Fig. 33E) in 96-well plates. Cell viability kinetics were captured on the IncuCyte system. NucGreen Dead diluted to 2 drops / mL was added to all wells containing cells with various culture medium components to capture the number of dead cells over time. Images were captured every 8 hours for 7 days without any intermittent feeding. The intensity of NucGreen Dead quantified the number of dead cells in the culture.

[0276] Functional characterization was assessed on WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia. Bacterial bioparticles labeled with pHrodo Red and pHrodo Red amyloid β were cryopreserved on day 23. WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia were cultured at 15,000–30,000 viable cells / cm³ three days after thawing. 2 Cells were plated at density in 96-well plates with 250 μl MMM (Fig. 33A-B) or supplemented with only MSCF (Fig. 33C-D) or IL-34 (Fig. 33E-F) or a combination of IL-34 and MCSF (Fig. 33G-H) in MDM matrix (AKA microglia basal medium). Cells were treated with diluted 1 μg / well conditioning or non-conditioning pHrodo bioparticles (Thermo Fisher #A10010, 2 mg per vial; stored at -20°C) (Fig. 33A, C, E, G) or pHrodo amyloid β (Fig. 33B, D, F, H). The plates were placed on IncuCyte and images of phagocytosis were taken at different time points up to 30 hours. WT and engineered microglia showed phagocytic function after thawing. The phagocytic kinetics and efficiency of WT, 1185 HT TREM2 KO, and 1187 HO TREM2 KO microglia differed.

[0277] The purity of wild-type (WT) microglia basal cells thawed on day 23 was determined in mature culture medium in the presence of MMM or supplemented with a combination of two key cytokines (IL-34, MSCF). Figure 35 Purity was quantified by harvesting cells on days 3, 7, and 14 post-thawing, and the purity of CD45, CD33, TREM2, CD11b, CX3CR1, P2RY12, TMEM119, and IBA was determined by harvesting cells at the end of differentiation and staining the cell surface and intracellular surfaces with markers using flow cytometry. Cryopreserved microglia maintained viability and purity in maturation medium supplemented with MSCF and IL-34. This simplified medium is invaluable for the co-culture of cryopreserved microglia with neurons and astrocytes to develop brain organoid models to study the contributions of numerous SNPs and mutations associated with neurodegeneration.

[0278] Example 8 - Generation of disease-associated microglia from patient-derived iPSCs

[0279] Recent genetic studies have shown that polymorphisms in several microglia-enriched genes are associated with altered risk of developing Alzheimer's disease (AD), Parkinson's disease (PD), and several neurodegenerative diseases. A list of risk-related SNPs summarizing GWAS studies from a cohort of end-stage microglia was generated from donors showing mutations in TREM2, CD33, and ABCA7 along with APOE isotypes. Cryopreserved microglia from patient-derived iPSCs provide an in vitro tool for creating more accurate models to understand the complex interactions between human microglia, neurons, and astrocytes in 2D or 3D organoid systems and to mimic neurodegenerative diseases (McQuade et al., 2019).

[0280] HPC generation from augmentatively reprogrammed AHNs and disease-specific iPSCs: E8 / MATRIGEL was used before collecting source material for differentiation toward hematopoietic cells and subsequently microglia. TMAdditional reprogrammed iPSCs generated from normal and disease-specific donors were adapted to hypoxia for at least 5-10 generations. A set of iPSC genotypes is described in Table 4. Karyotype analysis was performed on iPSCs from all donors, and the iPSC library was initiated for HPC differentiation using a 3D HPC differentiation protocol as described in Example 5. Microglia were generated by thawing cryopreserved HPCs and placing the cells in a 23-day differentiation process as described in Example 5. Cryopreserved microglia from different donors were thawed on day 23 and stained for the presence of microglia-specific markers. Cells were stained by flow cytometry for cell surface expression of CD45, CD33, TREM2, and CD11b, and intracellular expression of PU.1, IBA, P2RY12, TREM2, and TMEM119 proteins. Table 5 summarizes the purity obtained in all AHN and disease-associated microglia (DAM). The results showed that, without changing the differentiation protocol, high-purity microglia were generated from a group of healthy and disease-specific donors.

[0281] Table 4: Cryopreserved microglia produced from a group of epigenetically healthy (ANH) and disease-associated microglia (DAM) cells

[0282] APOE APOE APOE TREM2 CD33 Phenotype genotype gender rs429358 rs7412 genotype Rs7593268 Rs12459419 AHN N / A female T / T C / C 3 / 3 C / C T / T AHN N / A female T / T C / C 3 / 3 C / C T / T AHN N / A female T / T C / C 3 / 3 C / C T / T AHN TREM2 R47H female T / C C / T 3 / 3 C / T C / C AHN N / A male T / T C / C 3 / 3 C / C C / C AHN CD33 female T / C C / T 2 / 4 C / C T / T AD APOE 4 / 4 female C / C C / C 4 / 4 C / C C / C AD APOE 4 / 4 female C / C C / C 4 / 4 C / C C / C AD APOE 4 / 4 female C / C C / C 4 / 4 C / C C / T AD APOE 4 / 4 female C / C C / C 4 / 4 C / C T / T AHN ABCA7 G1527A male T / T C / C 3 / 3 C / C C / T AD APOE 2 / 4 female T / T C / C 2 / 4 C / C C / T

[0283] Table 5. Overview of purity of microglia epigenetic normal (ANH) and Alzheimer's disease (AD) donor samples.

[0284]

[0285] The levels of soluble TREM2 (sTREM2) protein secreted by microglia after thawing were quantified using a Simple Step ELISA (AbCam) from conditioned medium collected from a group of iPSC donors (Fig. 45). Microglia from epigenetic normal donors and disease-specific donors were thawed and plated at the same density in mature medium in 96-well Primaria plates. Used medium was collected on days 3 and 7 post-thawing. Cultures were fed semi-feeded with fresh mature medium on days 3 and 5 post-thawing.

[0286] Data revealed differences in soluble TREM2 levels among various samples of microglia derived from donors. Microglia from donors exhibiting the R47H genotype revealed the highest levels of soluble TREM on day 3 post-thawing and remained high even up to day 7 post-thawing. Despite being asymptomatic and thus classified as AHN, the iPSC-derived microglia secreted high levels of sTREM. This data is consistent with and correlates with high levels of sTREM2 observed in the cerebrospinal fluid of Alzheimer's disease patients (Cheng et al., 2016). This data provides a strong example of using iPSC-derived microglia to design predictive kits for screening the onset of neurodegenerative diseases. Younger donors with the APOE4 / 4 / genotype exhibiting AD onset showed higher levels of soluble TREM compared to older donors with the same genotype. The presence of SNPs in the CD33 or ABCA7 genes did not appear to enhance the release of soluble TREM from the culture supernatant. AHN donor 12068 showed high levels of soluble TREM at 3 and 7 days post-thaw.

[0287] Neuroinflammation contributes to the progression and pathogenesis of many neurodegenerative diseases. Resident microglia and astrocytes in the brain release cytokines that can exert pro-inflammatory and anti-inflammatory effects in the brain depending on stimulation and the microenvironment. This fluctuation between pro-inflammatory and anti-inflammatory signatures is associated with the onset of Alzheimer's disease (AD) and other neurodegenerative diseases. To quantify the levels of cytokines and chemokines released by disease-associated microglia (DAM), cryopreserved AHN and DAM microglia were thawed into MDM medium and seeded at 50,000 cells / well in Primaria 96-well plates. Cells were seeded for three days before starting stimulation with 100 ng / ml LPS to examine the M1-mediated response or stimulation with 10 ng / ml IL-4 and 10 μM dBu-cAMP to trigger an M2-specific response. Stimulation was performed in triplicate and lasted for 24 hours. The supernatant was centrifuged to remove cells and debris and immediately placed at -20°C. The supernatant was analyzed on a multiplex Luminex assay. The results of this multiple Luminex measurement were captured as a heatmap in Figure 27C.

[0288] Chemokines CCL1, CCL2, CCL3, CCL4, CCL8, CCL11, CCL13, CCL17, CCL18, CCL20, CCL22, and CCL24 act as chemokines and mediate the recruitment of myeloid cells, granulocytes, lymphoid cells, or neural progenitor cells to inflamed areas, enhancing phagocytic responses and typically upregulated in Alzheimer's disease (AD) or multiple sclerosis. In response to LPS or dBu-cAMP, microglia derived from APOE E4 / E4, AP0E E2 / E4, TREM2 R47H, ABCA7 G1527A, and CD33 (with the rs429358 SNP) release higher levels of all these analytes compared to the AHN lineage. The fold increase ranges from 0.1-fold to up to 7-fold across various microglia genotypes. This data from disease-associated microglia supports the early finding that CCL2 and CCL5 expression is increased in AD brain samples. Westin et al. have reported that CCL2 expression in the brain and cerebrospinal fluid (CSF) is a reliable predictor of AD severity.

[0289] Microglia derived from APOE E4 / E4, APOE E2 / E4, TREM2 R47H, ABCA7 G1527A, and CD33 (with the rs429358 SNP) release slightly elevated levels of soluble CD163, a marker of inflammation and inflammatory diseases associated with M2 polarization. Release of sCD163 prevents monocyte overactivation and reduces the secretion of pro-inflammatory cytokines TNF-α, IL-1β, IL-6, and IL-8. A similar trend was observed using chitinase-3, which plays a role in tissue remodeling during neuroinflammation (Melief et al., 2012; Minett et al., 2016).

[0290] PD-L1 and its receptor PD-1 trigger inhibitory signals that regulate the balance between T cell activation, tolerance, and immune-mediated tissue damage. In response to LPS, APOE E4 / E4-derived microglia were not increased compared to AHN-derived microglia. TREM2 R47H, APOE E2 / E4, and CD33 (with rs429358 SNP)-derived microglia showed an increase compared to the AHN lineage. In response to IL-4+dBu-cAMP, APOE E4 / E4, TREM2 R47H, and ABCA7 G1527A showed a slight increase compared to the AHN lineage, while APOE E2 / E4-derived microglia increased 7-fold compared to AHN-derived microglia.

[0291] Microglia derived from APOE E4 / E4, AP0E E2 / E4, TREM2 R47H, ABCA7 G1527A, and CD33 (with rs429358 SNP) release slightly elevated levels of soluble fractal chemokines, a soluble chemokine that promotes chemotaxis, survival, and enhanced neuroprotection by reducing TNF-α and nitric oxide levels during neuroinflammation.

[0292] APOE E4 / E4, APOE E2 / E4, and ABCA7 G1527A-derived microglia release high levels of CXCL1 / GROα in response to LPS and dBu-cAMP, while TREM2R47H and CD33-derived microglia show a slightly increased level of this cytokine release, suggesting a correlation between this analyte and the APOE and ABCA7 genotypes. A recent study suggested that CXCL1 may contribute to the inflammatory response in AD development but cannot serve as a potential genetic factor conferring susceptibility to AD in the pathogenesis of the disease. Under physiological conditions, CX3CR1 maintains microglial homeostasis by limiting microglial activation. The high level of this cytokine released after stimulation suggests the activation of a CX3CR1 rescue mechanism to maintain homeostatic function in disease-associated microglia related to the APOE or ABCA7 G1527A genotype. Alternatively, high levels of CX3CR1 secreted by microglia activated by APOEE4 / E4 and ABCA7 G1527A may be a signal that promotes neuronal degeneration (Atagi et al., 2015; Wolfe et al., 2018).

[0293] APOE E4 / E4 and APOE E2 / E4-derived microglia respond to LPS and IL4 / dBu-cAMP by releasing high levels of IL-6, while all other genotypes secrete IL-6 levels similar to those in AHN. IL-6 secretion attracts granulocytes, promotes cell-mediated humoral Th2 responses, and triggers inflammation. This mechanism would again support broader neuroinflammatory activity associated with the APOE E4 / E4 genotype.

[0294] ABCA7 G1527A-derived microglia respond to LPS by releasing high levels of IL-1β and IL-1α, while other genotypes secrete levels comparable to those of AHN microglia.

[0295] Compared to dBu-cAMP, APOE E4 / E4, AP0E E2 / E4, and ABCA7 G1527A-derived microglia also responded to LPS by releasing high levels of IL-8 / CXCL8. The high IL-8 release from ABCA7 G1527A-derived microglia suggests the initiation of a pro-inflammatory response that contributes to brain injury.

[0296] Microglia also secrete proteases and matrix metalloproteinases, which can eliminate Aβ deposition and limit AD processes, thus playing a neuroprotective role in AD. Compared with AHN-derived microglia, APOE E4 / E4, AP0E E2 / E4, TREM2 R47H, ABCA7 G1527A, and CD33 (with rs429358 SNP)-derived microglia release slightly elevated levels of MMP-9 and MMP-12.

[0297] When stimulated by LPS or M1, R47H TREM2-derived microglia released more than 7-fold IL-12p70 compared to AHN-derived microglia. Conversely, in response to IL4+dBu-cAMP or M2 stimulation, AP0E E2 / E4 microglia released more than 7-fold IL-12p70 compared to AHN-derived microglia. Other genotypes showed a slight increase in IL-12 secretion levels. Primary microglia produce IL-12 in the brain to control immune responses during infection or in Th1 cell-mediated autoimmune diseases of the CNS. The increased IL-12 levels in R47H TREM2-derived microglia imply a strong activation of NK cell and T cell cytotoxic activity.

[0298] Finally, microglia derived from APOE E4 / E4, AP0E E2 / E4, and ABCA7 G1527A released similar or slightly higher levels of IL-13, IL-18, IL-23, and α-synuclein, suggesting a correlation between the absence of these analytes and the aforementioned genotypes.

[0299] Neuroinflammation is a significant contributing factor to the pathogenesis and progression of Alzheimer's disease (AD). Combinations of several inflammatory mediators produce unique signatures associated with specific SNP mutations. iPSC-derived microglia from disease-specific donors can be used to identify key signature cytokines associated with various microglia SNPs and mutation-related genotypes.

[0300] Microglia's phagocytic function is crucial for maintaining neuroprotective effects. Microglia-mediated phagocytosis can be impaired by disease-specific SNPs or mutations, which can in turn affect key homeostatic mechanisms in the brain. This study assessed the phagocytic function of disease-associated microglia (DAMs) in the presence of pHrodo-labeled Staphylococcus aureus and amyloid-β to compare the effects of disease-associated SNPs on microglia's phagocytic function. This function can be used for high-throughput screening applications.

[0301] Among the disease-related genes expressed by these microglia, sequence variants in genes encoding trigger receptor 2 (TREM2) expressed on myeloid cells and the APO E isotype are associated with an abnormally increased risk of AD. APOE is the major cholesterol carrier in the brain and plays a crucial role in lipid transport, cholesterol homeostasis, and synaptic stability. Anti-ApoE immunotherapy inhibits the accumulation and deposition of amyloid protein, further supporting the role of ApoE in Aβ aggregation and clearance. ApoE expression has been shown to be significantly upregulated in disease-related microglia. The ApoE4 / E4 isotype has been shown to inherently affect the physiology of microglia by upregulating motor and phagocytic behavior in vitro. Reduced Aβ uptake has been shown with ApoE4 / E4 overexpression compared to other isotypes. The role of ApoE2 (the third most common major ApoE isotype) in delaying the onset of disease in familial AD has been shown in neurodegenerative diseases. To date, the isotype-specific effects of ApoE on iPSC-derived microglia function have not been thoroughly investigated.

[0302] TREM2 senses lipids and mediates myelin phagocytosis. Loss-of-function (LOF) variants of TREM2 are associated with increased amyloid plaque seeding, reduced amyloid aggregation, and interactions with increased ApoE trigger a signaling cascade leading to reduced microglia aggregation and ApoE accumulation in amyloid plaques, accompanied by functional impairment.

[0303] The phagocytic function of disease-associated microglia (DAMs) was evaluated in the presence of Staphylococcus aureus and amyloid-β to compare the effects of disease-associated SNPs on microglia phagocytic function.

[0304] Similarly, in genome-wide association studies, ATP-binding cassette transporter A7 (ABCA7) has been identified as a susceptibility factor for late-onset Alzheimer's disease. ABCA7 has been shown to mediate phagocytosis and affect membrane transport. ABCA7 is closely associated with AD. Phagocytic clearance of amyloid-β is impaired in Abca7- / - mice. Patient-derived iPSCs possess missense variants associated with the G1527A substitution in ABCA7, providing evidence that BCA7 plays a role in modulating Aβ homeostasis in the brain to alter phagocytic cell function.

[0305] CD33 is an immunomodulatory receptor associated with susceptibility to Alzheimer's disease (AD) by regulating phagocytosis in microglia. TREM2 interacts downstream of CD33 in regulating microglia physiology and metabolism; therefore, iPSC-derived microglia expressing WT and CD33 rs3865444 SNPs can be used to validate the role of CD33 in impaired phagocytic function (Caldeira et al., 2017).

[0306] Cryopreserved AHN and DAM microglia were thawed, cultured in maturation medium for three days, and exposed to pHrodo-labeled amyloid-β and pHrodo-labeled Staphylococcus aureus. Phagocytic kinetics were measured using the IncuCyte live-cell analysis system. Total red matter intensity was used to quantify functional responses.

[0307] Compared to AHN microglia, TREM2 R47H microglia and ABCA7-G1527A microglia exhibited strong phagocytic capacity against Staphylococcus aureus. CD33 microglia (with rs429358 SNP) showed comparable phagocytic capacity against Staphylococcus aureus to AHN microglia. TREM2 R47H microglia, ABCA7-G1527A microglia, and CD33 microglia (with rs429358 SNP) showed reduced phagocytic intensity in the presence of amyloid-β compared to AHN microglia.

[0308] Compared to AHN microglia, CW13030EE1 APOE 4 / 4 showed a stronger phagocytic activity against Staphylococcus aureus and amyloid β. Compared to AHN microglia, CW13098AA1 APOE 4 / 4 showed a weaker phagocytic activity against Staphylococcus aureus and amyloid β. Compared to AHN-derived microglia, CW13005AA1 APOE 2 / 4 microglia showed a strong phagocytic activity against amyloid β and a reduced phagocytic activity against Staphylococcus aureus. CW13074AA1 APOE 4 / 4 microglia showed a similar trend in phagocytosis against Staphylococcus aureus to AHN microglia, and a slightly enhanced phagocytic activity against amyloid β compared to AHN microglia.

[0309] A comprehensive understanding of genetic alterations targeting homeostatic, pro-inflammatory, and anti-inflammatory microglial subtypes can provide new biological insights and facilitate target prioritization for immunomodulatory therapies for neurodegenerative diseases.

[0310] Example 9 – Additional Characterization of Microglia

[0311] Extracellular nucleotides such as ATP and ADP are known to trigger receptor-mediated pathways known as purinergic signaling. Physiological processes such as tissue homeostasis, wound healing, neurodegeneration, immunity, inflammation, and cancer are regulated by purinergic signaling. Extracellular ATP and P2 receptors are important for microglia activation mechanisms. P2X receptors are ionotrophic receptors that bind to ATP or its derivatives. One of the P2Y receptors is a G protein-coupled receptor that responds to ADP. Under pathological conditions, nucleotides such as ATP are released or leaked from damaged cells and act as “find me” or “eat me” signals to induce processes such as chemotaxis and phagocytosis by microglia. P2 receptor activation also induces the production of cytokines from microglia, including interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNFα). These pro-inflammatory mediators have been shown to dynamically alter the expression and function of G protein-coupled receptors (GPCRs) in astrocytes.

[0312] The purinergic receptor response of microglia was characterized. Microglia were thawed in microglia differentiation medium and reconstituted into a cell suspension of 200,000 cells / well. 15 μl of microglia suspension was added to each well of a 384-well plate. Cells were treated with different doses (0-1000 nM) of B2ATP and ADP. AZ11645373 (P2X7 antagonist) and AZD1283 (P2Y) were also used. 12 The responses to BzATP and ADP were measured in the presence of potent receptor antagonists. For all treatments, 10 μL of 4X stock solution of the compound or inhibitor was added to the cells. Prior to assay, cells were exposed to the compound for 30 min in the presence or absence of the inhibitor. One vial of FLPR Calcium-6 was reconstituted to 11 mL with assay buffer B, and 15 μL of dye solution was added to the cell suspension in the presence of the compound. Cells were incubated at 37 °C for 1.5 h and imaged on an FDSS μCELL system.

[0313] Figure 44A Microglia with all traces of ATP / BzATP were shown. Figure 44B Microglia with ATP / BzATP sample traces are shown. Figure 44C -F shows the responses of microglia to BzATP, ADP, and BzATP in the presence of the P2X7 antagonist AZ11645373, and to BzATP in the presence of the P2X7 antagonist A438079. Figure 44G A dose-dependent response was demonstrated to demonstrate a functional ADP-dependent response in microglia in the presence of AZD1283.

[0314] Example 10 – Generating Neural Progenitor Cells from iPSCs

[0315] The successful development of in vitro disease models depends on the availability of a large number of late-stage lineages derived from patient-derived iPSCs. Neural progenitor cells (NPCs) are self-renewing progenitor cells with the ability to generate neurons and glial cells (Breunig et al., 2011). Many established protocols of varying efficiency exist for generating NPCs from primary neurons and iPSCs (Shi et al., 2012a, Shi et al., 2012b). Most recent protocols rely on the inhibition of the SMAD signaling pathway. This application describes a simple protocol to generate NPCs from different iPSC lineages by utilizing the spontaneous drift of iPSCs to the ectoderm without using dual SMAD inhibition pathways. The basic principle for generating this cell type is to pair it with iPSC-derived microglia to produce a long-term co-culture assay to mimic human brain development and the complex intercellular interactions between neural lineages, microglia, endothelial cells, pericytes, and astrocytes in discs derived from normal and disease-specific iPSC cells.

[0316] In this study, in MATRIGEL TM Multiple additionally reprogrammed iPSC lines were maintained on laminin- or hypotene-coated plates and E8 medium. Prior to differentiation, the iPSCs were kept under hypoxic conditions to generate neural progenitor cells. To initiate neural progenitor differentiation, iPSCs were harvested and cultured in E8 medium at 15 K / cm² in the presence of rock inhibitors. 2 Laying boards to MATRIGEL TM Cells were placed on laminin or hyalin plates. Cells were then placed in fresh E8 medium for the next 48 hours in the absence of rock inhibitors. The next step involved a pretreatment step, which involved placing the iPSC culture in DMEMF12 medium supplemented with 3 μM CHIR under normoxic conditions for 72 hours, changing the medium daily. At the end of the pretreatment step, cells were harvested and then plated at 30 K / cm². 2 Re-layout in 2D format on MATRIGEL TM 3D aggregates were generated at a density of 300,000 cells per milliliter on laminin or vitrin plates, or using ultra-low adhesion plates or rotating flasks in the presence of rock inhibitors. For the next 8 days, the cultures were fed every other day with N2-supplemented E6 medium under normoxic conditions. On day 14 of differentiation, the cultures were harvested and individualized using TrypLE. Cells were stained by cell surface staining for the presence of Tra-162, CD56, and CD15 for flow cytometry, and by intracellular staining for the presence of Sox1, neural epithelial stem cell protein, β3 microglobulin, and Pax-6 expression for flow cytometry. Different steps involved in NPC generation were described in... Figure 45A Overview. A summary of NPC markers appearing at different differentiation days in the three iPSC lineages. Figure 45B CD56 was used as a marker for NPCs obtained through this method. Cells were cryopreserved using CS10, and they maintained purity and proliferative potential upon thawing. NPCs were placed in downstream differentiation protocols to generate astrocytes and Pan neurons.

[0317] Following the protocol outlined by Julia et al., astrocytes were differentiated from NPCs. NPC cells on day 14 showed a growth rate of 15 kJ / cm². 2 Plated into MATRIGEL medium in Science Cell Astrocyte Medium. TM Coated 6-well plates. Perform a complete culture medium exchange on the plates every two days. Every 6 days, or when the culture is approximately 90% confluenced, harvest the plates using Accumax and dilute at 15 kJ / cm². 2 Re-lay boards onto MATRIGEL TM The cultures were coated onto 6-well plates. Cultures were fed and re-coated as described above for 4 passages. At the end of passage 4, the cultures were stained for surface markers CD44 and glutamate-aspartate transporter (GLAST) and intracellular markers glial fibrillary acidic protein (GFAP), excitatory amino acid transporter 1 (EAAT1), glutamine synthase (GS), aquaporin 4 (AQP4), and S100 calcium-binding protein B (S100β) (Fig. 45C).

[0318] Cortical glutamatergic neurons were generated from NPCs using a protocol developed by Slosarek et al. On day 14, NPCs were plated in E6 medium supplemented with 1 μM cyclic AMP, 10 ng / ml brain-derived neurotrophic factor (BDNF), and 10 ng / ml glial-derived neurotrophic factor (GDNF) for 30 days. The medium was then changed to cortical neural differentiation medium (E6 medium, 1 μM cyclic AMP, 10 ng / ml BDNF, 10 ng / ml GDNF, 100 ng / ml insulin-like growth factor-I, and 2% B27 supplement) for an additional 30 days (Brennan et al., 2011). Cortical glutamatergic neurons were observed between days 14 and 36 for different iPSC lines. The purity of the neural cultures was confirmed by staining for the presence of β3 tubulin and MAP2 expression.

[0319] ***

[0320] Based on this disclosure, all the methods disclosed and claimed herein can be made and carried out without excessive experimentation. While the compositions and methods of the present invention have been described according to preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the methods and steps or order of steps described herein without departing from the concept, spirit, and scope of the invention. More specifically, it will be apparent that certain chemically and physiologically relevant agents can be substituted for the agents described herein while obtaining the same or similar results. All such similar substitutions and modifications that will be apparent to those skilled in the art are considered to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0321] References

[0322] The following references are incorporated herein by reference to the extent that they provide exemplary procedures or supplement other details set forth herein.

[0323] Atagi et al., J Biol Chem. 290(43):26043-50, 2015.

[0324] Brennand et al., 2011

[0325] Caldeira et al., Front Aging Neurosci. 9:277, 2017.

[0326] Cheng et al., Clin Chim Acta. 463:88-95, 2016.

[0327] International Patent Publication No. WO 02 / 016536

[0328] International Patent Publication No. WO 03 / 016496

[0329] International Patent Publication No. WO 98 / 30679

[0330] International Patent Publication No. WO 98 / 53058

[0331] International Patent Publication No. WO 98 / 53059

[0332] International Patent Publication No. WO 98 / 53060

[0333] Julia et al., Stem Cell Reports. 9(2):600-614, 2017.

[0334] McQuade et al., J Mol Biol. 431(9):1805-17, 2019.

[0335] Melief et al., Glia. 60(10): 1506-17, 2012.

[0336] Minett et al., J Neuroinflammation. 13(1):135, 2016.

[0337] PCT Publication No. 2012 / 149484

[0338] Rustenhoven et al., Trends In Pharmacological Sciences, 38(3), 291-304, 2017.

[0339] Slosarek et al., Cell Rep. 24(9):2248-2260, 2018.

[0340] U.S. Patent Application No. 12 / 715,136

[0341] U.S. Patent No. 6,140,081

[0342] US Patent No. 6,453,242

[0343] US Patent No. 6,534,261

[0344] US Patent No. 6,617,152

[0345] US Patent No. 8,372,642

[0346] US Patent Publication No. 2002 / 0076747

[0347] US Patent Publication No. 2005 / 0064474

[0348] US Patent Publication No. 2005 / 0260186

[0349] US Patent Publication No. 2006 / 0104968

[0350] US Patent Publication No. 2006 / 0188987

[0351] US Patent Publication No. 2007 / 0218528

[0352] US Patent Publication No. 2011 / 0301073

[0353] US Patent Publication No. 2011 / 0301073

[0354] US Patent Publication No. 2011 / 0301073.

[0355] Westin et al., PLoS ONE.7:e30525, 2012.

[0356] Winkler et al., Brain pathology (Zurich, Switzerland), 24(4), 371–386, 2014.

[0357] Wolfe et al., Int J Mol Sci. 20(1), 2018.

Claims

1. An in vitro method for differentiation-induced pluripotent stem cells (iPSCs), comprising: (a) iPSCs were cultured on a positively charged amine surface under hypoxic conditions in the absence of extracellular matrix (ECM) to generate hematopoietic progenitor cells (HPCs). (b) Differentiation of HPCs into MSCs on a positively charged amine surface in a medium supplemented with ROCK inhibitors or blebbistatin; and (c) To generate pericytes by culturing MSCs on a positively charged surface in the absence of ECM in the presence of ECM.

2. The method of claim 1, wherein the iPSCs are cultured in a serum-free, composition-defined medium.

3. The method of claim 1, wherein the culture is carried out in the absence of laminin, fibronectin, hydrin, MATRIGEL™, tendinin, nestin, platelet-reactive protein, elastin, gelatin and / or collagen.

4. The method of claim 1, further comprising engineering the iPSC prior to step (a) to disrupt the expression of TREM2, SNCA, or MeCP2.

5. The method of claim 4, wherein the MSC is positive for CD73, CD44 and CD105.

6. The method of claim 5, wherein at least 90% of the differentiated MSCs are positive for CD73.

7. The method of claim 1, wherein the pericytes are positive for NG2, PDGFRβ and / or CD146.

8. The method of claim 1, wherein step (b) of the method does not include cell purification.

9. The method of claim 1, wherein the method conforms to Good Manufacturing Practices (GMP).

10. The method of claim 1, wherein the cultivation in step (a) is further defined as two-dimensional cultivation.

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