Method and device for obtaining amniotic mesenchymal stem cells from amniotic fluid and cells derived therefrom

Abstract

Methods for the purification, culture, and selection of mesenchymal stem cell (MSC) subsets with neonatal quality and adult tissue specificity for the production of advanced therapeutics.

Description

Technical Field

[0001] The present invention relates to methods for purifying, culturing and selecting a subpopulation of mesenchymal stem cells (MSCs) with neonatal quality tissue specificity for the production of advanced therapeutic drugs. Background Art

[0002] Amniotic fluid is the fluid that surrounds and protects the fetus during pregnancy. During the last trimester, amniotic fluid is secreted partly by the fetal lungs and partly by fetal urine. Amniotic fluid is ingested orally and absorbed by the fetal intestine, thereby re-entering the fetal circulation. Full-term amniotic fluid consists of water with electrolytes, but also contains proteins, carbohydrates, lipids, phospholipids, and urea. In addition to metabolic waste products, amniotic fluid also contains fetal cells and other materials shed from the skin, such as hair and vernix caseosa—a greasy deposit that covers the skin at birth. Tissue interfaces in contact with amniotic fluid contribute to the amniotic fluid's content, including cellular material. The lungs, the largest of these tissue interfaces, also secrete pulmonary surfactant into the TAF. The oral and nasal mucosa, the eyes, and the urinary tract are other such surfaces with non-keratinized epithelial interfaces in topological contact with amniotic fluid.

[0003] Mesenchymal stem cells (MSCs) are present in almost all tissues and are primarily located in the microenvironment around blood vessels. As will be appreciated by those skilled in the art, mesenchymal stem cells are multipotent stromal cells that can differentiate into a variety of cell types and also have anti-inflammatory, angiogenic properties for guiding tissue repair processes, making mesenchymal stem cells valuable for therapeutic treatment. The full-term amniotic fluid (TAF) collected during cesarean section contains many valuable cells, including MSCs. However, due to the difficulties associated with aseptic collection, processing TAF, and identification and extraction of MSCs, MSCs were not previously extracted and cultured on a large scale. In addition, specific MSC subpopulations may be particularly suitable for producing therapeutic drugs. In the past, MSCs derived from adult bone marrow, adult adipose tissue, or tissues associated with the birth of newborns (including placenta, umbilical cord, and cord blood) were widely used to obtain MSCs. Compared with MSCs derived from these adult sources, MSCs from these neonatal tissues may have additional abilities. In fact, several studies have reported biological properties superior to adult-derived MSCs, such as the increase in proliferation capacity, lifespan, and differentiation potential of MSCs from birth-associated tissues. However, none of these neonatal MSC sources have a corresponding tissue or organ in the adult body. Therefore, neonatal-quality MSCs with tissue specificity would be extremely beneficial. Furthermore, the acquisition of fetal material may be associated with negative effects on the infant. For example, in cord blood collection, it has been shown that as much cord blood as possible should be returned to the infant to improve survival, growth, and the development of fine motor skills. On the other hand, amniotic fluid is currently considered discarded medical waste. Therefore, the ethical and practical motivations for collecting this untapped resource are obvious. Summary of the Invention

[0004] Certain disclosed examples relate to devices, methods, and systems for obtaining amniotic mesenchymal stem cells from amniotic fluid, and cells derived therefrom. Those skilled in the art will appreciate that the applications of the devices, methods, and systems described herein are not limited to specific cell or tissue types. Further examples are described below.

[0005] In one aspect, the present disclosure provides a method for obtaining amniotic mesenchymal stem cells from amniotic fluid, comprising: providing term amniotic fluid (TAF); removing particulate matter from TAF to obtain purified TAF cells; subjecting the purified TAF cells to adhesion selection to obtain TAF adherent cells; passaging the TAF adherent cells to obtain TAF mesenchymal stem cells (TAF MSCs); and selecting TAF MSCs expressing a marker selected from the group consisting of: TBC1 domain family member 3K (TBC1D3K), allogeneic inflammatory factor 1-like (AIF1L), cadherin-related family member 1 (CDHR1), sodium / potassium transporting ATPase interacting protein 4 (NKAIN4), ATP binding cassette subfamily B member 1 (ABCB1), plasma membrane vesicle associated protein (PLVAP), mesothelin (MSLN), L1 cell adhesion molecule (L1CAM), hepatitis A virus cell receptor 1 (HAVCR1), mal, T cell differentiation protein 2 (gene / pseudogene) (MAL2), SLAM family member 7 (SLAMF7 ), double C2 domain β (DOC2B), endothelial cell adhesion molecule (ESAM), gamma-aminobutyric acid type A receptor β1 subunit (GABRB1), cadherin 16 (CDH16), immunoglobulin superfamily member 3 (IGSF3), desmoglein 3 (DSC3), regulator of hemoglobinization and erythroid expansion (RHEX), potassium voltage-gated channel interacting protein 1 (KCNIP1), CD70 molecule (CD70), GDNF family receptor α1 (GFRA1), Crumbs cell polarity complex component 3 (CRB3), tight junction protein 1 (CLDN1), novel transcript (AC118754.1), sodium voltage-gated channel alpha subunit 5 (SCN5A), fibroblast growth factor receptor 4 (FGFR4), potassium two-pore domain channel subfamily K member 3 (KCNK3), dysferlin (DYSF), ephrin A1 (EFNA1), potassium inward rectifier channel subfamily J member 16 (KCNJ16), membrane-associated ring-CH-type finger 1 (MARCHF1), synaptotagmin-like 1 (SYTL1), calponin 2 (CLSTN2), integrin subunit beta 4 (ITGB4), vesicle-associated membrane protein 8 (VAMP8), G protein-coupled receptor class C group 5 member C (GPRC5C), CD24 molecule (CD24), cadherin EGF LAG seven-transmembrane G-type receptor 2 (CELSR2), cadherin 8 (CDH8), glutamate receptor-interacting protein 1 (GRIP1), fasciclin actin-binding protein (DMTN), F11 receptor (F11R), cell adhesion molecule 1 (CADM1), cadherin 6 (CDH6), coagulation factor II thrombin receptor-like 2 (F2RL2), LY6 / PLAUR domain-containing protein 1 (LYPD1), solute carrier family 6 member 6 (SLC6A6), desmoglein 2 (DSG2), adhesion G protein-coupled receptor G1 (ADGRG1), cholecystokinin A receptor (CCKAR), oxytocin receptor (OXTR), integrin subunit alpha 3 (ITGA3), adhesion molecule with Ig-like domain 2 (AMIGO2), cadherin EGF LAG seven-transmembrane G-type receptor 1 (CELSR1), EPH receptor B2 (EPHB2).

[0006] In another aspect, the present disclosure provides isolated cells obtainable by the methods according to the present disclosure, the cells expressing surface markers selected from the group consisting of: TBC1 domain family member 3K (TBC1D3K), allogeneic inflammatory factor 1-like (AIF1L), cadherin-related family member 1 (CDHR1), sodium / potassium transporting ATPase interacting protein 4 (NKAIN4), ATP-binding cassette subfamily B member 1 (ABCB1), plasma membrane vesicle associated protein (PLVAP), mesothelin (MSLN), L1 cell adhesion molecule (L1CAM), hepatitis A virus cell receptor 1 (HAVCR1), mal, T cell differentiation protein 2 (gene / pseudogene) (M AL2), SLAM family member 7 (SLAMF7), double C2 domain β (DOC2B), endothelial cell adhesion molecule (ESAM), gamma-aminobutyric acid type A receptor β1 subunit (GABRB1), cadherin 16 (CDH16), immunoglobulin superfamily member 3 (IGSF3), desmoglein 3 (DSC3), regulator of hemoglobinization and erythroid expansion (RHEX), potassium voltage-gated channel interacting protein 1 (KCNIP1), CD70 molecule (CD70), GDNF family receptor α1 (GFRA1), Crumbs cell polarity complex component 3 (CRB3), tight junction protein 1 (CLDN1), novel transcript (AC118754.1), sodium voltage-gated channel alpha subunit 5 (SCN5A), fibroblast growth factor receptor 4 (FGFR4), potassium two-pore domain channel subfamily K member 3 (KCNK3), dysferlin (DYSF), ephrin A1 (EFNA1), potassium inward rectifier channel subfamily J member 16 (KCNJ16), membrane-associated ring-CH-type finger 1 (MARCHF1), synaptotagmin-like 1 (SYTL1), calponin 2 (CLSTN2), integrin subunit beta 4 (ITGB4), vesicle-associated membrane protein 8 (VAMP8), G protein-coupled receptor class C group 5 member C (GPRC5C), CD24 molecule (CD24), cadherin EGF LAG seven-pass transmembrane G-type receptor 2 (CELSR2), cadherin 8 (CDH8), glutamate receptor-interacting protein 1 (GRIP1), fasciclin actin-binding protein (DMTN), F11 receptor (F11R), cell adhesion molecule 1 (CADM1), cadherin 6 (CDH6), coagulation factor II thrombin receptor-like 2 (F2RL2), LY6 / PLAUR domain-containing protein 1 (LYPD1), solute carrier family 6 member 6 (SLC6A6), desmoglein 2 (DSG2), adhesion G protein-coupled receptor G1 (ADGRG1), cholecystokinin A receptor (CCKAR), oxytocin receptor (OXTR), integrin subunit alpha 3 (ITGA3), adhesion molecule with Ig-like domain 2 (AMIGO2), cadherin EGFLAG seven-pass transmembrane G-type receptor 1 (CELSR1), and EPH receptor B2 (EPHB2).

[0007] In certain embodiments, a method for obtaining term amniotic fluid mesenchymal stem cells (TAF MSCs) from term amniotic fluid may comprise:

[0008] Provide term amniotic fluid (TAF);

[0009] Removing particulate matter from TAF to obtain purified TAF cells;

[0010] Purified TAF cells were subjected to adhesion selection to obtain TAF-adherent cells;

[0011] passaging the TAF adherent cells to obtain a cell population comprising TAF MSCs; and

[0012] TAF MSCs are selected from the population as cells expressing at least one Group A surface marker, wherein the at least one Group A surface marker is selected from the group consisting of: TBC1 domain family member 3K, allogeneic transplant inflammatory factor 1-like, cadherin-related family member 1, sodium / potassium transporting ATPase interacting protein 4, ATP binding cassette subfamily B member 1, plasma membrane vesicle associated protein, mesothelin, L1 cell adhesion molecule, hepatitis A virus cell receptor 1, mal, T cell differentiation protein 2 (gene / pseudogene), SLAM family member 7, double C2 domain beta, endothelial cell adhesion molecule, gamma-aminobutyric acid type A receptor beta 1 subunit, cadherin 16, immunoglobulin superfamily member 3, desmocollin Mucin 3, regulator of hemoglobinization and erythroid cell expansion, potassium voltage-gated channel-interacting protein 1, CD70 molecule, GDNF family receptor α1, Crumbs cell polarity complex component 3, tight junction protein 1, novel transcript, sodium voltage-gated channel α subunit 5, fibroblast growth factor receptor 4, potassium two-pore domain channel subfamily K member 3, dysferlin, ephrin A1, potassium inward rectifier channel subfamily J member 16, membrane-associated RING-CH-type finger 1, synaptotagmin-like 1, calponin 2, integrin subunit β4, vesicle-associated membrane protein 8, G protein-coupled receptor class C group 5 member C, CD24 molecule, cadherin EGF LAG seven-transmembrane G-type receptor 2, cadherin 8, glutamate receptor-interacting protein 1, fasciclin actin-binding protein, F11 receptor, cell adhesion molecule 1, cadherin 6, coagulation factor II thrombin receptor-like 2, LY6 / PLAUR domain-containing protein 1, solute carrier family 6 member 6, desmoglein 2, adhesion G protein-coupled receptor G1, cholecystokinin A receptor, oxytocin receptor, integrin subunit α3, adhesion molecule with Ig-like domain 2, cadherin EGF LAG seven-transmembrane G-type receptor 1 and EPH receptor B2, thereby obtaining TAFMSCs.

[0013] In some instances, selecting TAF MSCs may comprise selecting TAF MSCs with reduced expression of a marker selected from the group consisting of IL13RA2, CLU, TMEM119, CEMIP, LSP1, GPNMB, FAP, CRLF1, MME, CLMP, BGN, DDR2. Removing particulate matter may comprise filtering and centrifuging TAF. Adhesion selection of purified TAF cells may comprise adhering the purified TAF cells to a surface coated with vitronectin. The selection step may be performed using fluorescence activated cell sorting (FACS). The selection step may be performed using antibodies against any marker or surface marker. The selection step may comprise selecting TAF MSCs expressing at least two markers from Group A surface markers. The selection step may comprise selecting TAF MSCs expressing at least three markers from Group A surface markers. The selection step may comprise selecting TAF MSCs expressing at least four markers from Group A surface markers. The selection step may comprise a plurality of sorting steps, each sorting step comprising directing the TAF MSCs into a first output group or a second output group based on a set of markers expressed or not expressed by the respective TAF MSCs.

[0014] In some examples, the selection step can comprise a first sorting step of directing TAF MSCs expressing group A surface markers to a first output group and a second sorting step of directing TAF MSCs from the first output group expressing a second group of markers to a second output group.

[0015] In certain examples, a method for obtaining term amniotic fluid-derived lung mesenchymal stem cells (TAF lung MSCs) from term amniotic fluid may comprise:

[0016] Provide term amniotic fluid (TAF);

[0017] Removing particulate matter from TAF to obtain purified TAF cells;

[0018] Purified TAF cells were subjected to adhesion selection to obtain TAF-adherent cells;

[0019] passaging the TAF adherent cells to obtain a cell population comprising TAF lung MSCs; and

[0020] TAF lung MSCs are selected from the population as cells expressing at least one Group B surface marker, wherein the at least one Group B surface marker is selected from the group consisting of: PCDH19, DDR1, MME, IFITM10, BGN, NOTCH3, SULF1, TNFSF18, BDKRB1, FLT1, PDGFRA, TNFSF4, UNC5B, FAP, CASP1, CD248, DDR2, PCDH18, LRRC38 and CRLF1, thereby obtaining TAF lung mesenchymal stem cells.

[0021] Selecting TAF lung MSCs may include excluding MSCs expressing markers selected from the group consisting of CD24, ITGB4, TNFSF10, GFRA1, CD74, FGFR4, HAVCR1, and OSCAR. The selection step may include selecting TAF MSCs expressing at least two surface markers from Group B surface markers. The selection step may include selecting TAF MSCs expressing at least three surface markers from Group B surface markers. The selection step may include selecting TAF MSCs expressing at least four surface markers from Group B surface markers. The selection step may include selecting TAF MSCs expressing surface markers selected from the group consisting of CD248, DDR1, and LRRC38. The selection step may include selecting TAF MSCs expressing CD248. The selection step may include selecting TAF MSCs expressing a combination of CD248 and a marker selected from the group consisting of DDR1 and LRRC38. The selection step may include selecting TAF MSCs expressing CD248, DDR1, and LRRC38. In some examples, isolated term amniotic fluid (TAF) mesenchymal stem cells can be obtained by the above method, wherein the cells express at least one group A surface marker.

[0022] In some examples, an isolated population of term amniotic fluid (TAF) mesenchymal stem cells can express at least one group A surface marker selected from the group consisting of: TBC1 domain family member 3K, allogeneic transplant inflammatory factor 1-like, cadherin-related family member 1, sodium / potassium transporting ATPase interacting protein 4, ATP binding cassette subfamily B member 1, plasma membrane vesicle associated protein, mesothelin, L1 cell adhesion molecule, hepatitis A virus cell receptor 1, mal, T cell differentiation protein 2 (gene / pseudogene), SLAM family member 7, dual C2 domain beta, endothelial cell adhesion molecule, gamma-aminobutyric acid type A receptor beta 1 subunit, cadherin 16, immunoglobulin Superfamily member 3, desmoglein 3, regulator of hemoglobinization and erythroid cell expansion, potassium voltage-gated channel-interacting protein 1, CD70 molecule, GDNF family receptor α1, Crumbs cell polarity complex component 3, tight junction protein 1, novel transcript, sodium voltage-gated channel α subunit 5, fibroblast growth factor receptor 4, potassium two-pore domain channel subfamily K member 3, dysferlin, ephrin A1, potassium inward rectifier channel subfamily J member 16, membrane-associated ring-CH-type finger 1, synaptotagmin-like 1, calponin 2, integrin subunit β4, vesicle-associated membrane protein 8, G protein-coupled receptor class C group 5 member C, CD24 molecule, cadherin EGF LAG seven-transmembrane G-type receptor 2, cadherin 8, glutamate receptor-interacting protein 1, fasciclin actin-binding protein, F11 receptor, cell adhesion molecule 1, cadherin 6, coagulation factor II thrombin receptor-like 2, LY6 / PLAUR domain-containing protein 1, solute carrier family 6 member 6, desmoglein 2, adhesion G protein-coupled receptor G1, cholecystokinin A receptor, oxytocin receptor, integrin subunit α3, adhesion molecule with Ig-like domain 2, cadherin EGF LAG seven-transmembrane G-type receptor 1, and EPH receptor B2.

[0023] In some instances, a composition may include a pharmaceutically acceptable carrier of the above-mentioned term amniotic fluid (TAF) mesenchymal stem cell population and TAF MSC separated. The term amniotic fluid (TAF) mesenchymal lung stem cells separated by the above method may express at least one group B surface marker, and the at least one group B surface marker is selected from the group consisting of: PCDH19, DDR1, MME, IFITM10, BGN, NOTCH3, SULF1, TNFSF18, BDKRB1, FLT1, PDGFRA, TNFSF4, UNC5B, FAP, CASP1, CD248, DDR2, PCDH18 and CRLF1. In certain instances, a group of term amniotic fluid (TAF) lung mesenchymal stem cells separated may express at least one group B surface marker.

[0024] In some examples, a method for obtaining term amniotic fluid renal mesenchymal stem cells (TAF renal MSCs) from term amniotic fluid may comprise:

[0025] Provide term amniotic fluid (TAF);

[0026] Removing particulate matter from TAF to obtain purified TAF cells;

[0027] Purified TAF cells were subjected to adhesion selection to obtain TAF-adherent cells;

[0028] passaging the TAF adherent cells to obtain a cell population comprising TAF renal MSCs; and

[0029] TAF renal MSCs are selected from the population as cells expressing at least one Group C surface marker, wherein the at least one Group C surface marker is selected from the group consisting of: HAVCR1, CD24, CLDN6, ABCB1, SHISA9, CRB3, AC118754.1, ITGB6, CDH1, LSR, EPCAM, AJAP1, ANO9, CLDN7, EFNA1, MAL2, F11R, L1CAM, GFRA1, IGSF3, TNF, MMP7, FOLR1, TGFA, C3, TNFSF10, PDGFB and WWC1, thereby obtaining TAF renal MSCs.

[0030] In certain instances, an isolated population of term amniotic fluid (TAF) renal mesenchymal stem cells (TAF renal MSCs) can express at least one Group C surface marker selected from the group consisting of: HAVCR1, CD24, CLDN6, ABCB1, SHISA9, CRB3, AC118754.1, ITGB6, CDH1, LSR, EPCAM, AJAP1, ANO9, CLDN7, EFNA1, MAL2, F11R, L1CAM, GFRA1, IGSF3, TNF, MMP7, FOLR1, TGFA, C3, TNFSF10, PDGFB, and WWC1.

[0031] A composition may comprise a population of term amniotic fluid (TAF) renal mesenchymal stem cells isolated as described above.

[0032] In some examples, a method for obtaining term amniotic fluid skin mesenchymal stem cells (TAF skin MSCs) from term amniotic fluid may comprise:

[0033] Provide term amniotic fluid (TAF);

[0034] Removing particulate matter from TAF to obtain purified TAF cells;

[0035] Purified TAF cells were subjected to adhesion selection to obtain TAF-adherent cells;

[0036] passaging the TAF adherent cells to obtain a cell population comprising TAF skin MSCs; and

[0037] selecting TAF skin MSCs from the population as cells expressing at least one Group D surface marker selected from the group consisting of TNFSF18, PCDH19, NCAM2, TNFSF4, CD248, DDR2, HTR2B, PCDH18, SULF1, MME, ADGRA2, DCSTAMP, PDGFRA, UNC5B, SCUBE3, CEMIP, BDKRB1, FLT1, BDKRB2, FAP, CASP1, and SRPX2; and

[0038] Obtain TAF skin MSCs.

[0039] In certain instances, an isolated population of term amniotic fluid (TAF) skin mesenchymal stem cells (skin MSCs) can express at least one Group D surface marker selected from the group consisting of TNFSF18, PCDH19, NCAM2, TNFSF4, CD248, DDR2, HTR2B, PCDH18, SULF1, MME, ADGRA2, DCSTAMP, PDGFRA, UNC5B, SCUBE3, CEMIP, BDKRB1, FLT1, BDKRB2, FAP, CASP1, and SRPX2. A composition can comprise the isolated population of term amniotic fluid (TAF) skin mesenchymal stem cells and a pharmaceutically acceptable carrier for TAF skin MSCs.

[0040] In some examples, a method for obtaining term amniotic fluid neural mesenchymal stem cells (TAF neural MSCs) from term amniotic fluid may comprise:

[0041] Provide term amniotic fluid (TAF);

[0042] Removing particulate matter from TAF to obtain purified TAF cells;

[0043] Purified TAF cells were subjected to adhesion selection to obtain TAF-adherent cells;

[0044] passaging the TAF adherent cells to obtain a cell population comprising TAF neural MSCs; and

[0045] TAF neural MSCs are selected from the population as cells expressing at least one Group E surface marker, wherein the at least one Group E surface marker is selected from the group consisting of: HAVCR1, ACKR3, OSCAR, C3, SIRPB1, SLC6A6, CCKAR, TNFSF10, CLSTN2, TENM2, SFRP1, PIK3IP1, SCNN1D, CLDN11, ALDH3B1 and ITGB4, thereby obtaining TAF neural MSCs.

[0046] In some examples, an isolated population of term amniotic fluid (TAF) neural mesenchymal stem cells (TAF neural MSCs) can express at least one Group E surface marker selected from the group consisting of: HAVCR1, ACKR3, OSCAR, C3, SIRPB1, SLC6A6, CCKAR, TNFSF10, CLSTN2, TENM2, SFRP1, PIK3IP1, SCNN1D, CLDN11, ALDH3B1, and ITGB4. A composition can include the isolated population of term amniotic fluid (TAF) neural mesenchymal stem cells and a pharmaceutically acceptable carrier for TAF neural MSCs.

[0047] In certain aspects, the present disclosure provides methods and apparatus for isolating term amniotic fluid (TAF) mesenchymal stem cells and compositions comprising TAF mesenchymal stem cells comprising one or more features of the foregoing description and / or drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 is a flow chart showing the steps for purification, culture and selection of MSC subsets.

[0049] Figure 2 This diagram shows how to collect amniotic fluid.

[0050] Figure 3 is a schematic diagram of a perspective view of an apparatus for filtering amniotic fluid according to an example.

[0051] Figure 4 is a schematic diagram of a cross-sectional side view of an apparatus for filtering amniotic fluid according to an example.

[0052] Figure 5 is a schematic diagram of a cross-sectional side view of an apparatus for filtering amniotic fluid according to an example.

[0053] Figure 6 is a schematic diagram of a cross-sectional side view of an apparatus for filtering amniotic fluid according to an example.

[0054] Figure 7 is a schematic diagram of a cross-sectional side view of an apparatus for filtering amniotic fluid according to an example.

[0055] Figure 8 is a schematic diagram of a cross-sectional side view of an apparatus for filtering amniotic fluid according to an example.

[0056] Figure 9 is a schematic diagram of a cross-sectional side view of an apparatus for filtering amniotic fluid according to an example.

[0057] Figure 10 is a schematic diagram of a cross-sectional side view of an apparatus for filtering amniotic fluid according to an example.

[0058] Figure 11 is a schematic diagram of a cross-sectional side view of an apparatus for filtering amniotic fluid according to an example.

[0059] Figure 12 a is a schematic diagram of a cross-sectional side view of an apparatus for filtering amniotic fluid according to an example.

[0060] Figure 12 b is a device for filtering amniotic fluid according to an example Figure 10 Schematic diagram of section AA in a.

[0061] Figure 13 is a flow chart of a method of filtering amniotic fluid according to an example.

[0062] Figure 14 is a flow chart illustrating the steps for calculating the MSC tissue-specific score according to an example.

[0063] Figure 15 is a graph showing examples of MSC tissue-specific scores representing 5% and 15% thresholds.

[0064] Figure 16 is an example graph showing tissue prioritized data and tissue distal data, including tissue prioritized data greater than the 15th percentile.

[0065] 17A to 17D Shown are the results of an example study demonstrating the effect of using TAF lung MSCs to treat rats with induced pulmonary fibrosis. DETAILED DESCRIPTION

[0066] Methods for purifying, culturing, and selecting MSC subsets with neonatal quality and adult tissue specificity are summarized in Figure 1 The examples disclosed herein relate to devices and methods for collecting, purifying, isolating, expanding, differentiating, and maturing amniotic fluid-derived cells. The examples disclosed herein are not limited to collecting a certain type of amniotic fluid-derived cell, and the techniques disclosed herein are broadly applicable to different cells and tissues.

[0067] Amniotic fluid collection

[0068] Amniotic fluid may be collected to produce term amniotic fluid (TAF) according to the methods described in U.S. Patent Application No. 14 / 776,499 (corresponding to US2016 / 0030489), the entire contents of which are incorporated herein by reference. Figure 2 is a block diagram of an example of an amniotic fluid collection method 300 according to an exemplary embodiment of the present invention. It should be understood that the method 300 may include any number of additional or alternative tasks. Figure 3 The tasks shown in FIG300 do not need to be performed in the order depicted, and method 300 may be incorporated into a more comprehensive process or procedure having additional functionality not described in detail herein.

[0069] like Figure 2 As shown, method 300 may include, for example, making an incision 301 in the uterine wall of a pregnant woman during a cesarean section. Step 301 may be performed using a standard physician's scalpel. Figure 2 As shown, method 300 may include inserting an amniotic fluid collector 302 through the incision in the uterine wall made in step 301. Method 300 also includes penetrating the amniotic membrane 303 using the amniotic fluid collector of step 302. Step 303 may also include penetrating the chorion. In one aspect, the tip is inserted to a depth of 10 cm. In some instances, the tip is inserted to a depth of about 3 cm to about 30 cm. In some instances, the tip is inserted to a depth of about 4 cm, about 5 cm, about 6 cm, about 7 cm, about 8 cm, about 9 cm, about 10 cm, about 11 cm, about 12 cm, about 13 cm, about 14 cm, about 15 cm, about 16 cm, about 17 cm, about 18 cm, about 19 cm, about 20 cm, about 21 cm, about 22 cm, about 23 cm, about 24 cm, about 25 cm, about 26 cm, about 27 cm, about 28 cm, or about 29 cm.

[0070] Method 300 also includes collecting amniotic fluid from the amniotic sac using the amniotic fluid collector of step 302 304. Step 304 can include activating a siphon to transfer the amniotic fluid to a collection chamber of the amniotic fluid collector, such as by opening an inlet valve of the amniotic fluid collector. Step 304 can also include positioning the collection chamber of the amniotic fluid collector below the inlet of the amniotic fluid collector. Step 304 can also include coupling a negative pressure source to an outlet of the amniotic fluid collector to initiate the transfer of the amniotic fluid. Step 304 can include repositioning the inlet of the amniotic fluid collector to recover substantially all of the available amniotic fluid.

[0071] Finally, method 300 includes removing the amniotic fluid collector from the amniotic sac 905. Step 905 can include closing the inlet valve of the amniotic fluid collector. In one example, no blood is visible in the collected material. Step 905 can also include emptying the collection system for further use / processing and sterilizing the exterior of the entire device. In one example, the exterior is sterilized using 70% ethanol to maintain sterility during any post-processing steps, such as separation of cellular material and fluid storage according to the present invention, such as in a laminar flow hood facility.

[0072] In one example, the amniotic fluid collection procedure is completed in less than one minute. In one example, the amniotic fluid collection procedure is completed in 1 to 2 minutes. In one example, the amniotic fluid collection procedure is completed in no more than 3 minutes. In one example, the method is simplified compared to standard operating procedures for cesarean sections, for example by preventing amniotic fluid from spilling into the surgical wound, thereby improving visibility and physical access. In one example, fetal skin is not affected by the tip of the device.

[0073] purification

[0074] Term amniotic fluid (TAF) is purified by filtering term amniotic fluid to remove vernix caseosa. Although the term "term amniotic fluid" is used herein and elsewhere in this disclosure, it should be understood that the methods, processes and devices of the present disclosure can be applied to all amniotic fluids, not just term amniotic fluid. Term amniotic fluid can be amniotic fluid collected at the time of a full-term cesarean section delivery using, for example, a closed catheter-based system. For the purposes of this specification, "term amniotic fluid" can be amniotic fluid collected at a planned cesarean section after 37 completed weeks of gestation or later, or amniotic fluid collected at near-term, for example, at a planned cesarean section after 36 completed weeks of gestation. Preferably, term amniotic fluid is collected at the time of a planned cesarean section at 37 completed weeks of gestation or later.

[0075] Figure 3is a schematic diagram of an apparatus 100 for filtering amniotic fluid according to one example. Amniotic fluid contains amniotic cells, such as mesenchymal stem cells, that originate from the fetus or the amniotic sac. Amniotic fluid also contains other materials shed from the skin, such as hair and vernix caseosa. Materials other than amniotic cells are referred to herein as particulate matter and may also include meconium, blood clots, etc. Particulate matter can be considered to be any material larger than 20 μm. For filtering purposes, it may be particularly advantageous to treat any material larger than 30 μm or even 50 μm as particulate matter. Optionally, any material larger than the target amniotic cell can be treated as particulate matter. Amniotic fluid therefore typically contains a mixture of amniotic cells and particulate matter. Apparatus 100 comprises a filter 101 for filtering particulate matter from amniotic fluid and a chamber 102 enclosing filter 101. Chamber 102 comprises a fluid inlet 103 and a fluid outlet 104. Enclosing the chamber 102 of the filter 101 should be interpreted as isolating the filter 101 from the environment surrounding the chamber 102 by the chamber, such that there is no fluid communication between the amniotic fluid in the chamber 102 and the environment. Figure 3 In the example of , fluid communication through the chamber 102 is thus controlled via the fluid inlet 103 and the fluid outlet 104. The filter 101 is connected to the interior of the chamber 102 between the fluid inlet 103 and the fluid outlet 104. Figure 12 The circular chamber 102 and the filter 101 are shown as follows Figure 12 However, it should be understood that the chamber 102 and the filter 101 may have different shapes to optimize for different applications. The apparatus 100 includes an inlet connector 105 that is arranged to connect between the fluid inlet 103 and the amniotic fluid sample source 201 (e.g., Figure 4 A sealed connection is formed between the two. Figure 4 A schematic example of such a source 201 is shown. An inlet connector 105 is connected to the fluid inlet 103 and configured to provide a sealed connection directly between the fluid inlet 103 and the amniotic fluid source 201, minimizing exposure to contaminants and effectively sterilizing the amniotic fluid. This facilitates obtaining amniotic cells that can be processed post-filtration to improved quality standards. Thus, sterile pharmaceutical production processes are facilitated. For example, the preparation of surfactant molecules can be facilitated. Apparatus 100 is used to improve the function of amniotic stem cells, such as improved engraftment after transplantation. Such improved processes are achieved by enclosing the filter 101 within the chamber 102 and arranging the inlet connector 105 to form a sealed connection between the fluid inlet 103 of the chamber 102 and the amniotic fluid sample source 201. Thus, the risk of amniotic stem cells being exposed to contaminants such as bacteria and viruses is reduced. Exposure to oxygen is also minimized, which reduces the formation of oxygen free radicals that can negatively impact stem cell function.

[0076] Figure 31 shows an example where the inlet connector 105 includes a tube 105 connected to the fluid inlet 103 at a first sealing connection 114. The inlet connector 105 can form a sealed connection with the fluid inlet 103 by a force fit connection, adhesive, clamp or other fixing element. In another example, as Figure 4 As schematically shown in FIG, the inlet connector 105 is a continuous extension of the fluid inlet 103 and does not require a separate fixing element, but is formed as a single piece, for example by molding or other material forming techniques. Figure 3 and Figure 4 A second connector 115 is shown which is configured to form a sealed connection with a sample source 201, such as a container or bag 201 containing amniotic fluid. The second connector 115 may comprise a releasable force fit connection, a clamp, or a combination thereof or other releasable securing element. The chamber 102, the filter 101, the fluid inlet 103, the fluid outlet 104 and the inlet connector 105 may be provided in a sterile package as a kit, such as a disposable kit. Such a kit, i.e. the apparatus 100, therefore provides a convenient and improved process for filtering and obtaining amniotic stem cells. Thus, in use, as the amniotic fluid flows from the fluid inlet 103 to the fluid outlet 104, the amniotic fluid passes through the filter 101. Particulate matter is thus deposited on the filter 101 and the amniotic fluid containing the amniotic cells flows through the fluid outlet 104. As Figure 12 As shown in the example of FIG, filter 101 can be connected to the inner wall 113 of chamber 102 around its edge 116. This prevents unfiltered amniotic fluid from flowing from inlet 103 to outlet 104. Filter 101 can be tensioned or otherwise supported to prevent folding or bending of filter 101 within chamber 102. This maintains a defined mesh size or pore size over the entire area of ​​filter 101, and thus, defined filtering properties. Maintaining a defined mesh size or pore size also reduces the risk of clogging filter 101. This can improve long-term performance.

[0077] The apparatus 100 may include an outlet connector 106 to form a sealed connection between the outlet and an amniotic cell receiving device 202 , such as a centrifuge or other amniotic cell processing equipment downstream of the apparatus 100 . Figure 4 A schematic example of such an apparatus 202 is shown. This minimizes exposure to contaminants and enables effective sterilization of the amniotic fluid during post-filtration processing steps. Figure 3 1 shows an example in which the outlet connector 106 includes a tube 106 connected to the fluid outlet 104 at a first sealing connection 117. The outlet connector 106 can form a sealed connection with the fluid outlet 104 by a force fit connection, adhesive, clamp or other fixing element. In another example, as Figure 4As schematically shown in FIG, outlet connector 106 is a continuous extension of fluid outlet 104 and does not require a separate fixing element, but is formed as a single piece, such as by molding or other material forming techniques. Figure 3 and Figure 4 A second connector 118 is shown, configured to form a sealed connection with an amniotic cell processing device downstream of the apparatus 100, such as the centrifuge 202. The second connector 118 can include a force-fit connection, a clamp, a combination thereof, or other releasable securing element. Thus, the connection between the second connector 118 and, for example, the centrifuge 202 can be repeatedly connected and disconnected, and can also be resealed to maintain a sealed connection during such processing. The chamber 102, filter 101, fluid inlet 103, fluid outlet 104, inlet connector 105, and outlet connector 106 can be provided in sterile packaging as a kit, such as a disposable kit. This kit, i.e., the apparatus 100, provides a convenient and improved process for filtering and processing amniotic stem cells. The apparatus 100 can include pumps 122, 123, which are arranged to pressurize the amniotic fluid to flow from the fluid inlet 103 to the fluid outlet 104. This allows for more efficient filtration of the amniotic fluid, allowing larger volumes of amniotic fluid to be filtered in a shorter period of time.

[0078] Figure 6 An example is shown in which a pump 122 is connected to the fluid outlet 104 to draw amniotic fluid in the direction of the arrow shown through the filter 101. The pump 122 can be arranged at the fluid inlet 103 to push the amniotic fluid through the filter 101. The pump 122 can be a small, manually operated pump that is integrated with the fluid inlet 103, the fluid outlet 104, the inlet connector 105, or the outlet connector 106.

[0079] Figure 7 Another example of a pump 123 arranged to pressurize amniotic fluid to cause it to flow from the fluid inlet 103 to the fluid outlet 104 is shown, as will be described in more detail below. The chamber 102 can include a conduit 119 disposed between the fluid inlet 103 and the fluid outlet 104. The pressure in the chamber 102 can vary in response to fluid and / or gas communication through the conduit 119. The flow rate of amniotic fluid through the filter 101 can thus be optimized depending on the application, for example, by increasing or decreasing the flow rate through the filter 101 by varying the pressure in the chamber 102 via the conduit 119.

[0080] Figure 5 An example of a conduit 119 communicating with the chamber 102 is shown. An access port 120, such as a connector or valve element, can be actuated to allow fluid or gas to be exhausted from the chamber 102 and / or injected into the chamber 102 to affect the pressure therein. Figure 5In the embodiment, the conduit 119 is arranged between the fluid outlet 103 and the filter 101, but in another example, the conduit 119 can be arranged between the fluid inlet 103 and the filter 101. Figure 5 A further example of a conduit 119 in communication with the chamber 102 is shown. A pump 123 may be arranged in communication with the conduit 119, such as Figure 7 This is advantageous in optimizing the flow rate in chamber 102 and the associated filtration process. Figure 7 In the example of , the conduit 119 is in variable communication with the upstream cavity 108 of the chamber 102 and the downstream cavity 109 of the chamber 102 , that is, the filter 101 can be arranged to divide the chamber 102 into the upstream cavity 108 and the downstream cavity 109 . Figure 7 In the embodiment, the conduit 119 is connected to both the upstream cavity 108 and the downstream cavity 109. The pump 123 is arranged to pressurize the amniotic fluid so that the amniotic fluid flows from the upstream cavity 108 to the downstream cavity 109, or from the downstream cavity 109 to the upstream cavity 108. The latter case may be advantageous in cases where a momentary reverse flow is desired, for example, to clear a blockage or obstruction of the filter 101. In this case, as shown in FIG. Figure 7 Valves 120, 120', 121, 121' schematically shown are operated to provide the desired flow direction. For example, for reverse flow, valves 120 and 121' can be open, while valves 120' and 121 can be closed. In normal filtration mode, valves 121, 121' can be open, while valves 120, 120' can be closed. In this filtration mode, upstream chamber 108 can also be pressurized by opening valve 120'.

[0081] The filter 101 may include a first filter element 101a and a second filter element 101b disposed between the first filter element 101a and the fluid outlet 104, as shown in FIG. Figure 8 As shown schematically. The mesh size or pore size of the second filter element 101b can be smaller than the mesh size or pore size of the first filter element 101a. This can effectively filter particles of gradually smaller sizes. Therefore, the risk of filter blockage is reduced. This makes the filtering process of amniotic fluid more reliable and robust. An improved method for filtering amniotic fluid containing particles of a larger particle size range is also provided. Furthermore, since stem cells will not be lost in blocked pores, a larger proportion of stem cells in the amniotic fluid can be obtained. Although Figure 8Two filter elements 101a and 101b are shown in FIG. 1 , but it should be understood that any number of filter elements can be arranged in the chamber 102 in a sequence of gradually decreasing mesh sizes or pore sizes in the direction of fluid flow from the fluid inlet 103 to the fluid outlet 104 to effectively filter particles of gradually decreasing sizes. The first filter element 101a and the second filter element 101b can be separated by a certain distance (d) along the direction of amniotic fluid flow from the fluid inlet 103 to the fluid outlet 104, as shown in FIG. Figure 8 The movement of amniotic fluid between the first filter element 101a and the second filter element 101b, which in some cases may involve turbidity flow, can further reduce the risk of unwanted accumulation of particles on the first and second filter elements 101a, 101b.

[0082] The filter 101 may comprise a mesh having a mesh size in the range of 20 to 2000 μm. In another example, the filter 101 comprises a mesh having a mesh size in the range of 100 to 500 μm. This can be particularly effective in filtering particulate matter from the amniotic fluid. Figure 8 , the first filter element 101a can comprise a mesh with a mesh size ranging from 500 to 1000 μm, and the second filter element 101b can comprise a mesh with a mesh size ranging from 30 to 150 μm. The first filter element 101a can thus remove larger debris, followed by the second filter element 101b to remove smaller particles. This enables particularly effective filtration of particles of varying sizes, further reducing the risk of clogging, and reliable filtration of particles of increasing volume over a longer period of time. As previously mentioned, any number of filter elements can be arranged serially in chamber 102.

[0083] Figure 9 Three filter elements 101a, 101b, 101c are shown arranged in the chamber 102. In some examples, the filter element with the smallest mesh size or pore size arranged most downstream in the chamber 102, such as Figure 6 The filter element 101b and Figure 9Filter element 101c in the filter 101 may have a specific mesh size or pore size so that only single amniotic cells or amniotic cell clumps of less than 10 cells pass through filter 101. In such an example, the minimum mesh size or pore size may be approximately 30 μm. Filter 101 may comprise a mesh, such as a nylon mesh. Filter 101 may comprise a porous material having a variable pore size in the direction of amniotic fluid flow through filter 101 from fluid inlet 103 to fluid outlet 104. That is, larger debris is removed at the surface of filter 101 closest to inlet 103, while smaller particles are removed deeper into the filter as amniotic fluid flows through filter 101 toward outlet 104 and the pore size decreases. As previously described, chamber 102 may comprise an upstream chamber 108 and a downstream chamber 109. Upstream chamber 108 and downstream chamber 109 may be formed as a single piece to form chamber 102, for example, by molding or other material forming techniques. The upstream cavity 108 and the downstream cavity 109 may be formed as separate units which are then connected to each other, for example, by adhesive or welding, to form a sealed connection. The filter 101 may be connected simultaneously or sequentially using such a welding process or by the aforementioned adhesive.

[0084] The upstream cavity 108 and the downstream cavity 109 may be releasably connected to each other at a connecting element 110 to form a sealed connection, such as Figure 9 This enables the chamber 102 to be opened, for example to replace the filter 101. The filter 101 can thus be releasably connected to the chamber 102, for example Figure 7 In the embodiment of the present invention, the filter elements 101a, 101b, 101c can be releasably connected to the chamber 102. This allows for convenient customization for different applications, as filter elements 101a, 101b, 101c with different pore or mesh sizes or different numbers of such filter elements can be installed in the chamber 102.

[0085] The connecting element 110 is configured to form a sealed connection between the upstream cavity 108 and the downstream cavity 109 and may include an annular gasket extending around the edge of the upstream cavity 108 and the downstream cavity 109. The filter 101 may include a filter cartridge consisting of different numbers of filter elements 101a, 101b, 101c having different pore sizes that can be customized for a specific amniotic fluid sample. For example, the evaluation of amniotic fluid turbidity and milkiness (the level of fetal fat in terms of particle size and opacity) can be used as an indicator for using an appropriate filter cartridge. An accompanying chart to which the amniotic fluid sample is compared can indicate which filter cartridge to use. The upstream cavity 108 and / or the downstream cavity 109 may be funnel-shaped. Figures 3 to 9 An example is shown in which both the upstream cavity 108 and the downstream cavity 109 are funnel-shaped. Figure 11An example is shown in which only the downstream cavity 109 is funnel-shaped. Having a funnel shape may be advantageous for directing the flow of amniotic fluid along a desired symmetric vector through the filter 101 and the apparatus 100. The upstream cavity 108 and / or the downstream cavity 109 may comprise cavity walls 111a, 111b that are arranged substantially parallel to the filter 101, i.e., perpendicular to the direction of flow of the amniotic fluid from the fluid inlet 103 to the fluid outlet 104. Figure 10 An example is shown in which the chamber walls 111a, 111b of the upstream chamber 108 and the downstream chamber 109 are arranged substantially parallel to the filter 101. This minimizes the space within the chamber 102 while maintaining sufficient filtration area to minimize the risk of air introduction, which could interfere with surfactant molecules, reduce the risk of infection, and reduce the harmful formation of reactive oxygen species in the amniotic cells. The chamber 102 and / or the inlet connector 105 and / or the outlet connector 106 can be formed from phthalate-free PVC material. This provides a device suitable for contacting pharmaceutical starting materials such as amniotic cells.

[0086] The apparatus 100 may include a protrusion 112 disposed extending from an inner wall 113 of the chamber 102 . Figure 11 and Figure 12 Examples of such protrusions 112 are shown in a cross-sectional side view and through section AA, respectively. The protrusions 112 provide support for the filter 101, preventing the filter 101 from beginning to bend and fold towards the inner wall 113. Thus, in this case, flow through the mesh or pores of the filter 101 is still possible because the filter 101 can be supported by the protrusions 112 at a distance from the inner wall 113, i.e., the protrusions 112 can further limit the risk of throttling and provide efficient, robust, and reliable filtration.

[0087] Figure 13 1 is a flow chart of a method 300 for filtering amniotic fluid containing particulate matter and amniotic cells. The method 300 comprises forming a sealed connection 301 between a fluid inlet 103 of a chamber 102 and an amniotic fluid sample source 201. The method 300 comprises passing 302 the amniotic fluid through a filter 101 enclosed in the chamber 102 by providing a flow of amniotic fluid from the fluid inlet 103 to a fluid outlet 104 of the chamber 102. Particulate matter is thereby deposited on the filter 101, and the amniotic fluid containing the amniotic cells flows through the outlet 104. The method 300 thus provides a method as described above with respect to the apparatus 100 and Figures 3 to 12 Advantageously, the method 300 provides for efficient and sterile filtration of amniotic fluid to obtain high-quality amniotic cell samples.

[0088] In one embodiment, the removal of particulate matter from TAF to obtain purified TAF cells can be accomplished by applying any method known in the art, such as filtration, centrifugation, etc. TAF can be filtered through a filter with a pore size equal to or greater than 20 μm. The filter can be made of any synthetic material, including but not limited to cellulose acetate, cellulose nitrate (collodion), polyamide (nylon), polycarbonate, polypropylene, and polytetrafluoroethylene (Teflon). In one embodiment, the removal of particulate matter is accomplished by applying apparatus 100.

[0089] Adhesion Selection

[0090] Various terms well known to those skilled in the art have been and will be used throughout the specification sheets, for example, the term "expression (express, expression and / or expressing)" in the context of cell surface markers refers to the presence of specific markers on the cell surface, and the surface markers have been produced by cells.Surface marker expression can be used to select between different cell populations, for example, the positive selection indication of surface marker expression selects a cell population that is more strongly expressed than another cell population of specific surface markers. On the contrary, the negative selection indication of cell surface marker expression selects a cell population that is more weakly expressed than another cell population of specific surface markers.

[0091] As described above and elsewhere in the specification, TAFs contain various progenitor cell types. In certain instances, specific progenitor cell types can be isolated and propagated via adhesion selection. For example, vitronectin substrates, Synthemax (Merck, II-SC substrate, CLS3535-1EA) can be used as a coating to create a more in vivo-like environment for stem cell culture, thereby limiting the maturation of TAF-derived progenitor cells and maintaining plasticity. Synthemax is an animal-free, synthetic, flexible vitronectin-based peptide substrate for serum or serum-free expansion of human progenitor / stem cells and other adult stem cell types. Those skilled in the art will understand that the vitronectin-based peptide substrate may include a portion of vitronectin, such as a specific peptide sequence of vitronectin. Alternatively, intact vitronectin may be used. Synthemax vitronectin substrate provides a synthetic, xeno-free alternative to biocoatings and / or feeder cell layers commonly used in cell culture and known in the art. Briefly, a standard tissue culture treated flask can be coated with approximately 0.2 mL of Synthemax / cm at 10 μg / mL. 2 , producing 2 μg / cm 2and incubated at 37°C for about 1 hour, 1.5 hours, 2 hours, 4 hours, 8 hours, or more than 8 hours, or at room temperature for about 2 hours, 1 hour, 4 hours, 8 hours, or more than 8 hours, wherein the remaining solution is optionally removed and replaced. In certain examples, Synthemax can be coated at a surface density of about 1 to 5 μg / cm 2 , such as 2μg / cm 2 , 0.1 to 10 μg / cm 2 , 0.5 to 4 μg / cm 2 , 1 to 3 μg / cm 2 or about 1.5 to 2.5 μg / cm 2 .

[0092] In other embodiments, adhesion selection can be performed using a surface coated with, for example, collagen, fibronectin. Alternatively, adhesion selection can be performed using an uncoated surface comprising tissue culture treated plastic.

[0093] The cells purified from TAF can be gently resuspended in pre-warmed xeno-free cell culture medium, and the cell suspension is then added to the Synthemax coated flask. The culture medium can be changed once at different times after being added to the flask, for example, after about 2 hours to 168 hours, 12 hours to 96 hours, 24 hours to 72 hours, 36 hours to 60 hours, 42 hours to 56 hours or 48 hours, and then changed to about: every day, every other day, every three days, every five days, once a week, once every two weeks or about less than once every two weeks. By repeatedly removing the spent culture medium, unattached cells can be removed, thus selecting MSC by its affinity for being attached to the surface processed through Synthemax. Cell culture can be for a period of time, such as about 4 days, 7 days, 10 days, 11 days, 12 days, 13 days, 14 days, 18 days, 21 days, 28 days or more than 21 days. Optionally, in some instances, cells can be cultured under hypoxic conditions, and hypoxic activation can change cell metabolism during amplification, increase resistance to oxidative stress, thereby improving the implantation of the transplanted MSC, survival in the ischemic microenvironment, and angiogenesis potential. After cultivation, the P0 colonies (colony forming units-CFU) that have been formed can be separated and pooled. After pooling, the remaining cells may be mainly non-tissue specific MSCs. In certain instances, the pooled P0 cells can be gently resuspended in a pre-warmed xeno-free cell culture medium and then plated in a tissue culture flask without Synthemax treatment for passage. The pooled cells can be inoculated at a seeding density between about 100 and 10,000 cells / cm 2 , 500 to 8000 cells / cm 2 1000 to 5000 cells / cm 2or approximately 2000 to 4000 cells / cm 2 The culture medium can be changed approximately every 1 day, 2 days, 4 days, or more than 4 days. After a period of time, such as about 2 days, 4 days, 7 days, or more than 7 days, the cells can be dissociated and harvested. Further selective MSC isolation can be achieved as described below.

[0094] Identification of markers

[0095] When the gene expression profiles of TAF-MSCs derived from adipose tissue or bone marrow were compared with those of adult MSCs by RNAseq, TAF-MSCs tended to express more of some genes present in adult MSCs and less of others. The identification of positive and negative TAF-MSC-specific neonatal cell surface markers enables the use of ligands such as antibodies and aptamers or other selection techniques to sort neonatal-quality MSCs from those that have further differentiated and are less important as progenitor cells.

[0096] Cell surface markers that distinguish tissue-associated cells from other MSCs can be elucidated via a bioinformatics approach using a tissue-specific scoring algorithm. An example of a MSC tissue-specific scoring algorithm is shown in Figure 14 Tissue specificity can be determined as a combination of two components: "tissue transcriptional similarity" also referred to as a similarity score and "tissue-specific gene expression program" also referred to as a gene set score. In some instances, the similarity score can be the average Spearman correlation with each MSC tissue reference sample (e.g., fetal lung MSC sample). In an example, the gene set score can be the average expression level of genes in the tissue-specific gene set. Figure 14 As shown, in certain examples, after normalizing the similarity and gene set scores using a Z-transform to convert the input values, which are real or complex sequences, into a complex frequency domain representation, and then combining them, assigning equal weight to each score, and transforming the combined values ​​using a Z-transform, the resulting output is an MSC tissue-specificity score. The MSC tissue-specificity score measures the relative tissue specificity between input samples by determining how many standard deviations of a sample are more or less specific for a given tissue compared to the average input sample. For example, the MSC tissue-specificity score can indicate how many clone samples appear to have a tissue-specific phenotype, such as a lung phenotype. This approach can identify the top X% percentile scores using a normal distribution function, effectively identifying the top X% of clones that are most tissue-specific for the tissue of interest.

[0097] In one example, for a particular tissue, a tissue-prioritized clone can be defined as any clone that falls within the top X% percentile score, where X is any percentage within a range from about 0.1 to 25, such as about 1, 5, 10, 15, and 20, and from about 30 to 75, such as about 35, 40, 45, 50, 55, 60, 65, or 70. Examples of TAF-MSC tissue-specific prioritization results are shown in Figure 15 , where thresholds of 15% and 5% are visible. Tissue-specific clones are prioritized and candidate surface marker genes can then be identified. For each tissue, two groups can be defined: a tissue-prioritized group and a tissue-distal group. This assay can be performed using a suitable analysis program, such as DEseq2 from Bioconductor.org. The tissue-prioritized group can include clones scoring in the top 15% percentile. The tissue-distal group can include clones scoring in the bottom Y% percentile, where Y is any percentage within a range, the lower limit of the range being from about 25 to 70, such as about: 30, 35, 40, 45, 50, 55, 60 or 65 and the upper limit being from 75 to 99.9, such as about: 80, 85, 90, 95 or 99. Figure 16 An example of such analysis is shown for kidney tissue. Next, genes differentially expressed between the tissue prioritization group and the tissue distal group can be identified. Finally, the differential expression results can be annotated with surface marker gene information.

[0098] In certain examples, to identify tissue-specific cell surface markers, the surface marker genes have a greater than Z-fold increase, wherein the Z-fold increase is at least about: a 1.5-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 5-fold, 8-fold, 10-fold, 12-fold, 15-fold or even more fold increase (log2FoldChange) expressed by the prioritized clones compared to the average clones and can be selected to be greater than about 500, such as greater than about: 1000, 1500, 2000, 2500, 3000, 5000 or even greater Transcripts Per Kilobase Million (TPM) to give the top ranked tissue-specific marker candidates, such as about the top: 5, 10, 20, 30, 40, 50, 60, 70, 100 or more, such as those shown in Tables 3 to 6 below and described in more detail below. Appropriate log2FoldChange and TPM values ​​may vary even further, depending on tissue type specificity and on the abundance / absence of good markers.

[0099] Applying the above-described tissue-specific algorithm to identify surface markers, after adherence selection and passaging, TAF-MSCs may express various surface markers identified as shown in Table 1 below, indicating non-tissue-specific TAF-MSCs. Those skilled in the art will appreciate that such surface markers may be present at various surface densities and may be upregulated or downregulated compared to other cell types. Therefore, such surface markers can be used to identify and isolate specific cell types. In some cases, the surface markers listed in Table 1 below may be expressed at an average high level of at least 8-fold higher in TAF-MSCs compared to other MSC cell types, particularly adult MSCs derived from bone marrow or adipose tissue. The thresholds used to generate Table 1 are as follows: X was selected as 15%, Y was selected as 50%, Z was selected as 8-fold, and TPM was selected as greater than 3000. Those skilled in the art will appreciate that the numbers used in Table 1 and all tables herein are intended only to indicate the total number of markers identified and do not indicate that a particular marker is more strongly and / or more preferentially expressed than another.

[0100] 1.TBC1D3K TBC1 domain family member 3K

[0101] 2.AIF1L Allogeneic transplant inflammatory factor 1-like

[0102] 3.CDHR1 cadherin-related family member 1

[0103] 4.NKAIN4 Sodium / potassium transporter ATPase interacting protein 4

[0104] 5.ABCB1 ATP-binding cassette subfamily B member 1

[0105] 6.PLVAP plasma membrane vesicle associated protein

[0106] 7.MSLN Mesothelin

[0107] 8.L1CAM L1 cell adhesion molecule

[0108] 9.HAVCR1 Hepatitis A virus cellular receptor 1

[0109] 10.MAL2 mal, T cell differentiation protein 2 (gene / pseudogene)

[0110] 11.SLAMF7 SLAM family member 7

[0111] 12.DOC2B double C2 domain β

[0112] 13. ESAM endothelial cell adhesion molecule

[0113] 14.GABRB1 γ-aminobutyric acid type A receptor β1 subunit

[0114] 15.CDH16 Cadherin 16

[0115] 16.IGSF3 Immunoglobulin superfamily member 3

[0116] 17.DSC3 Desmosome Glucocortin 3

[0117] 18.RHEX Hemoglobinization and Erythroid Cell Expansion Regulator

[0118] 19.KCNIP1 Potassium voltage-gated channel interacting protein 1

[0119] 20.CD70 CD70 molecule

[0120] 21.GFRA1 GDNF family receptor α1

[0121] 22.CRB3 Crumbs cell polarity complex component 3

[0122] 23.CLDN1 tight junction protein 1

[0123] 24.AC118754.1 novel transcript

[0124] 25.SCN5A sodium voltage-gated channel alpha subunit 5

[0125] 26.FGFR4 Fibroblast growth factor receptor 4

[0126] 27.KCNK3 Potassium two-pore domain channel subfamily K member 3

[0127] 28.DYSF dysferlin

[0128] 29.EFNA1 Ephrin A1

[0129] 30.KCNJ16 Potassium inward rectifier channel subfamily J member 16

[0130] 31.MARCHF1 membrane-associated ring-CH-type finger 1

[0131] 32.SYTL1 Synaptotagmin-like 1

[0132] 33.CLSTN2 Calcineurin 2

[0133] 34.ITGB4 integrin subunit β4

[0134] 35.VAMP8 vesicle-associated membrane protein 8

[0135] 36.GPRC5C G protein-coupled receptor class C group 5 member C

[0136] 37.CD24 CD24 molecule

[0137] 38.CELSR2 Cadherin EGF LAG seven-transmembrane G-type receptor 2

[0138] 39.CDH8 Cadherin 8

[0139] 40.GRIP1 Glutamate receptor interacting protein 1

[0140] 41.DMTN Fascin actin binding protein

[0141] 42.F11R F11 receptor

[0142] 43.CADM1 cell adhesion molecule 1

[0143] 44.CDH6 Cadherin 6

[0144] 45.F2RL2 Coagulation factor II thrombin receptor-like 2

[0145] 46.LYPD1 LY6 / PLAUR domain-containing protein 1

[0146] 47.SLC6A6 Solute carrier family 6 member 6

[0147] 48.DSG2 Desmoglein 2

[0148] 49.ADGRG1 Adhesion G protein-coupled receptor G1

[0149] 50.CCKAR cholecystokinin A receptor

[0150] 51.OXTR Oxytocin receptor

[0151] 52.ITGA3 integrin subunit α3

[0152] 53.AMIGO2 Adhesion molecule with Ig-like domain 2

[0153] 54.CELSR1 Cadherin EGF LAG seven-transmembrane G-type receptor 1

[0154] 55.EPHB2 EPH receptor B2

[0155] Table 1

[0156] As will be appreciated by those skilled in the art, suitable combinations of the markers listed in Table 1 can be used to isolate TAF MSCs from adult MSCs by selecting specific markers from Table 1 or a combination of two, three, four, five, six, or more markers from Table 1. In certain instances, TAF MSCs can be more specifically identified compared to adult MSCs by identifying a combination of markers that are more strongly expressed, for example, 8-fold or more, of any combination of the aforementioned markers, such as TBC1D3K and / or AIF1L and / or CDHR1 and / or NKAIN4 and / or ABCB1 and / or PLVAP. When a combination of markers is used, identification can be achieved using a lower threshold of stronger expression, such as 2-fold or more, 4-fold or more, or 6-fold or more, of each of the markers.

[0157] In contrast to the above surface markers that may be expressed more strongly on the surface of TAF-MSCs compared to adult MSCs (positive markers), in certain instances, the following surface markers in Table 2: IL13RA2, CLU, TMEM119, CEMIP, and LSP1 may be expressed more weakly on TAF-MSCs compared to other cell types (negative markers), such as when any combination of the aforementioned markers is expressed at 1 / 8-fold or less relative to adult MSCs (optionally, a TPM threshold of >500). When using a combination of negative markers, identification can be achieved using a lower threshold of weaker expression, such as 1 / 2-fold or less, 1 / 4-fold or less, or 1 / 6-fold or less of each of the markers.

[0158] Combinations of two or more of these negative markers can also be used to more specifically isolate TAF MSCs. In addition, those skilled in the art will also recognize that combinations comprising both negative and positive markers, such as at any of the above thresholds, can also effectively and more specifically isolate TAF MSCs.

[0159] 1.IL13RA2 Interleukin-13 receptor subunit α2

[0160] 2. CLU

[0161] 3. TMEM119 transmembrane protein 119

[0162] 4. CEMIP cell migration induction hyaluronidase 1

[0163] 5.LSP1 Lymphocyte-specific protein 1

[0164] 6.GPNMB Glycoprotein Nmb

[0165] 7. FAP Fibroblast activation protein alpha

[0166] 8.CRLF1 Cytokine receptor-like factor 1

[0167] 9.MME Membrane Metalloendopeptidase

[0168] 10.CLMP CXADR-like membrane protein

[0169] 11.BGN biglycan

[0170] 12.DDR2 discoidin domain receptor tyrosine kinase 2

[0171] Table 2

[0172] Marker-based selection

[0173] Amniotic fluid contains a uniform fluid containing heterologous cells. Therefore, it may be necessary to select based on markers. An example of selection based on markers is by means of fluorescence activated cell sorting (FACS). Fluorescence activated cell sorting (FACS) can be used to purify the cell population of TAF-MSCs. Even when the target cell type expresses very low levels of identification markers and / or needs to be separated based on the difference in marker density, FACS can also obtain the desired cell population with very high purity. FACS can purify individual cells based on size, granularity and fluorescence. As will be understood by those skilled in the art, FACS can be used to select certain cell populations that express a cell surface marker more than another cell population, and vice versa. In some examples of purification methods, a large number of purification methods such as panning, complement consumption and magnetic bead separation can be used in combination with FACS or as a substitute for FACS. In short, in order to purify the target cells by means of FACS, they are first stained with fluorescently labeled monoclonal antibodies (mABs) that recognize specific surface markers on the desired cell population. The reverse selection of unstained cells can also achieve separation. For GMP production of cells according to some embodiments, FACS can use closed system sorting technology such as Run. The sample can be kept contamination-free in the disposable, fully enclosed MACSQuant Tyto chamber (MACSQuantTyto Cartridge). In addition, filtered air can drive cells through the microchannel into the microchip at very low pressure (<3PSI). However, before entering the microchannel, potential cell clumps can be trapped by the filter system, thereby ensuring a smooth sorting process. The fluorescence detection system can detect target cells based on predetermined fluorescence parameters of the cells. Based on their fluorescence and scattered light characteristics, the target cells can be redirected by the sorting valve located in the microchannel. For certain examples of purification methods, the success of staining and the resulting sorting depends largely on the selection of identification markers and the selection of mAbs. Sorting parameters can be adjusted according to purity and yield requirements. Unlike conventional droplet sorters, cells sorted by MACSQuant Tyto may not be subjected to high pressure or charge, nor may they be decompressed. Therefore, this gentle sorting method may result in high cell activity and functionality. Alternatively, other marker-based selection techniques are known to those skilled in the art and are used herein. These selection techniques include, but are not limited to, magnetic activated cell sorting, microfluidics-based sorting, buoyancy-activated cell sorting, mass cytometry, and the like.

[0174] Tissue-specific cells and uses

[0175] TAF lung cell marker

[0176] As described above, analysis of RNAseq data and publicly available expression data sets from TAF-MSC clones, adult and neonatal MSC reference materials, and fetal fibroblasts can be used to identify and characterize TAF-MSC cells. For example, TAF-MSC subpopulations can be established by clustering their expression data (RNAseq) with neonatal reference samples. Such subpopulations include, but are not limited to, lung MSCs, urinary tract MSCs (also described as kidney MSCs in this disclosure), and skin MSCs. A gene list of highly expressed and lowly expressed genes for each cluster of expression data enables the identification of surface marker genes for each cluster. Using such data comparisons, TAF cell subpopulations are compared with adult MSC cells based on their gene expression (RNAseq) to derive a list of neonatal-specific surface marker genes for each cluster. Many target surface markers associated with lung TAF cells have been identified. For example, a non-exclusive list of preferred surface markers for identifying and separating TAF lung cells is provided below. In addition, due to the limited number of different MSC subtypes in TAF, the selection of tissue-specific MSCs can first be accomplished by characterization, followed by a stepwise reverse selection / sorting of the material by considering the combined (multivariate) surface marker profiles of different tissue-specific MSCs. Those skilled in the art will appreciate that any such combination of these surface markers can be used to identify and isolate lung TAF cells from a general TAF-derived cell and / or TAF-MSC cell population. In some instances, the following non-exclusive list of surface markers can be expressed more highly on the surface of TAF lung cells compared to other cell types, such as other TAF-derived cells and / or TAF-MSC cells.

[0177] As described above, bioinformatics techniques can be used to identify tissue-specific surface markers, and thus, expression of the surface markers identified in Table 3 may be increased at least 10-fold on prioritized clones compared to common TAF-MSC clones (optionally, TPM threshold > 2000).

[0178] 1. PCDH19- protocadherin 19;

[0179] 2.DDR1-discoid domain receptor tyrosine kinase 1;

[0180] 3.MME-membrane metalloendopeptidase;

[0181] 4.IFITM10-interferon-induced transmembrane protein 10;

[0182] 5.BGN-diglycosides;

[0183] 6. NOTCH3-Notch receptor 3;

[0184] 7.SULF1-sulfatase 1;

[0185] 8.TNFSF18-TNF superfamily member 18;

[0186] 9.BDKRB1-bradykinin receptor B1;

[0187] 10.FLT1-fms-related tyrosine kinase 1;

[0188] 11.PDGFRA-platelet-derived growth factor receptor alpha;

[0189] 12.TNFSF4-TNF superfamily member 4;

[0190] 13.UNC5B-unc-5 spindle protein receptor B;

[0191] 14.FAP-fibroblast activation protein alpha;

[0192] 15.CASP1-caspase 1;

[0193] 16.CD248-endosialin;

[0194] 17.DDR2-discoid domain receptor tyrosine kinase 2;

[0195] 18. PCDH18 - Protocadherin 18; and / or

[0196] 19.CRLF1-cytokine receptor-like factor 1;

[0197] Table 3

[0198] In contrast to the above surface markers that may be expressed more strongly on the surface of TAF lung MSCs, in certain instances, the following surface markers: CD24, ITGB4, TNFSF10, GFRA1, CD74, FGFR4, HAVCR1, and OSCAR may be expressed more weakly on TAF lung MSCs compared to other cell types, such as other TAF-derived cells and / or TAF-MSCs. As will be appreciated by those skilled in the art, one, two, three, four, or more of the above surface markers that are expressed more weakly can be used to isolate TAF lung cells from other cell types, such as other TAF-derived cells and / or TAF-MSCs.

[0199] In certain instances, the cell surface marker CD248 (endosialin) can be used to sort TAF lung MSCs from a TAF MSC population. Further surface markers that can be used to sort TAF lung MSCs include DDR-1 (discoidin domain receptor tyrosine kinase 1) and LRRC38 (leucine-rich repeat-containing protein 38), all three of which have been identified as useful markers for isolation via antibodies. In some instances, endosialin, DDR-1, and / or LRRC38 alone or in combination with other markers can be used for sorting. Endosialin can be combined with DDR-1 or LRRC38 for sorting, or DDR-1 and LRRC38 can be combined without endosialin.

[0200] As will be appreciated by those skilled in the art, suitable combinations of the markers listed in Table 3 with CD248, DDR-1, and LRR38 can be used to isolate TAF lung MSCs from TAF MSCs by selecting specific markers from Table 3 or a combination of two, three, four, five, six, or more markers and / or CD248 and / or DDR-1 and / or LRR38 from Table 3. In certain instances, TAF lung MSCs can be more specifically identified compared to TAF MSCs by identifying a combination of markers that are more strongly expressed, for example, 10-fold or more stronger expression (optionally, a TPM threshold of >2000) of any combination of the aforementioned markers, such as PCDH19 and / or DDR1 and / or MME and / or IFITM10 and / or BGN and / or NOTCH3 and / or CD248 and / or DDR-1 and / or LRR38. When a combination of markers is used, identification can be achieved using a lower threshold of stronger expression, such as 4-fold or more, 6-fold or more, or 8-fold or more expression of each marker.

[0201] In contrast to the above surface markers that can be expressed more strongly on the surface of TAF lung MSCs compared to TAF MSCs (positive markers), in certain instances, the following surface markers: CD24, ITGB4, TNFSF10, GFRA1, CD74, FGFR4, HAVCR1, and OSCAR can be expressed more weakly on TAF lung MSCs compared to other cell types (negative markers), such as 1 / 8-fold or less expression of any combination of the above markers relative to TAF MSCs (optionally, TPM>500). When using a combination of negative markers, identification can be achieved using a lower threshold of weaker expression, such as 1 / 2-fold or less, 1 / 4-fold or less, or 1 / 6-fold or less expression of each of the markers.

[0202] Combinations of two or more of these negative markers can also be used to more specifically isolate TAF lung MSCs. In addition, those skilled in the art will also recognize that combinations including both negative and positive markers, such as at any of the above thresholds, can also effectively and more specifically isolate TAF lung MSCs.

[0203] 17A to 17D Shown is an example of the results of a proof-of-principle study on the potential use of TAF lung MSCs for treatment, which was performed using newly sorted TAF MSCs expressing MSC lung cell surface markers, including CD248, DDR1, and LRRC38 (referred to as "LBX-THX-001 cells"). The purpose of this study was to investigate the effect of LBX-THX-001 cells in a bleomycin-induced male rat model of pulmonary fibrosis. Two cell concentrations (2M cells / kg and 5M cells / kg) and two types of vehicles for the cells (PBS and CryoStor CS-10) were tested.

[0204] The development of pulmonary fibrosis in rats following exposure to bleomycin is well documented in the literature and is a common model used to study the pathology of pulmonary fibrosis and the effects of various treatments. The number of LBX-THX-001 cells injected was chosen to be relevant to potential human therapy. Therefore, the number of cells was chosen to reflect the number of cells used in previous studies in rats (8 to 20 M cells / kg) and humans (0.5 to 2 M cells / kg).

[0205] Bleomycin (every 1000U / rat) is instilled into 34 male SD rat trachea for inducing rat pulmonary fibrosis.During the first week, rats are monitored and weighed every day, thereafter twice a week until the study terminates.On the 4th day after bleomycin attack, LBX-THX-001 cells are administered by intravenous (iv) injection.The injection volume is 194 to 535 μ L (maximum tolerated injection volume 1mL / kg).Based on the previous experience with the model, the reaction to the bleomycin intratracheal instillation is as expected, and the model loses weight on the first day after instillation and during the recovery period thereafter.The weight loss between the bleomycin group and the treatment group does not have significant differences.

[0206] like 17A to 17D As shown, bleomycin instillation induced fibrotic changes in the lung. Histopathological evaluation inferred the pathological changes in the bleomycin group in terms of both the percentage of parenchyma affected and the modified Ashcroft scale score. 17A to 17D As shown, the group treated with LBX-THX-001 cells (2 million cells / kg) 4 days after bleomycin had significantly less fibrosis in their lungs compared to the bleomycin group. This was demonstrated using the readout "percent parenchyma affected" ( 17A to 17B) and fibrosis score modified Ascot scale ( 17A to 17D No human MSCs were detected in the rat lungs at the end of the experiment (after 28 days).

[0207] TAF renal cell marker

[0208] Similar to the TAF lung MSC cell marker identified above, many target surface markers associated with kidney TAF cells have been identified. For example, a non-exclusive list of surface markers for identifying and separating TAF kidney MSC is provided in Table 4 below. Similar to the TAF lung MSC marker, the expression of the surface markers identified in Table 4 on prioritized kidney TAF clones can be increased by at least 12 times (optionally, TPM threshold value>2000) compared to common TAF-MSC clones. In addition, due to the limited number of different MSC subtypes in TAF, the selection of tissue-specific MSC can first be by characterization, and then by considering the combination (multivariate) surface marker spectrum of different tissue-specific MSCs, material is progressively reversed / sorted to complete. It will be appreciated by those skilled in the art that any such combination of these surface markers can be used for identifying and separating TAF kidney cells from general TAF derived cells and / or TAF-MSC cell populations. In some instances, compared to other cell types, such as other TAF derived cells and / or TAF-MSC cells, the non-exclusive list of the following surface markers can be expressed higher on the surface of TAF kidney cells:

[0209] 1.HAVCR1-Hepatitis A virus cellular receptor 1;

[0210] 2.CD24-CD24 molecule;

[0211] 3.CLDN6-tight junction protein 6;

[0212] 4.ABCB1-ATP-binding cassette subfamily B member 1;

[0213] 5. SHISA9-shisa family member 9;

[0214] 6.CRB3-Crumbs cell polarity complex component 3;

[0215] 7.AC118754.1 - Arachidonic acid 15-lipoxygenase, ALOX15, Smoothelin-like protein 2, SMTNL2, glutathione hydrolase 6, GGT6, Myb binding protein 1A, MYBBP1A, protein homolog 2, SPNS2

[0216] 8.ITGB6-integrin subunit β6;

[0217] 9.CDH1-cadherin 1;

[0218] 10.LSR-lipolysis-stimulating lipoprotein receptor;

[0219] 11.EPCAM-epithelial cell adhesion molecule;

[0220] 12.AJAP1-adherens junction associated protein 1;

[0221] 13.ANO9-anoctamin 9;

[0222] 14.CLDN7-tight junction protein 7;

[0223] 15.EFNA1-ephrin A1;

[0224] 16.MAL2-mal, T cell differentiation protein 2 (gene / pseudogene);

[0225] 17.F11R-F11 receptor;

[0226] 18.L1CAM-L1 cell adhesion molecule;

[0227] 19.GFRA1-GDNF family receptor α1;

[0228] 20.IGSF3-immunoglobulin superfamily member 3;

[0229] 21.TNF-tumor necrosis factor;

[0230] 22.MMP7-matrix metalloproteinase 7;

[0231] 23.FOLR1-folate receptor alpha;

[0232] 24.TGFA-transforming growth factor alpha;

[0233] 25.C3-complement C3;

[0234] 26.TNFSF10-TNF superfamily member 10;

[0235] 27. PDGFB - platelet-derived growth factor subunit B; and / or

[0236] 28.WWC1 - WW and C2 domain-containing protein 1.

[0237] Table 4

[0238] As will be appreciated by those skilled in the art, suitable combinations of the markers listed in Table 4 can be used to isolate TAF renal cells from TAF-MSCs by selecting specific markers from Table 4 or a combination of two, three, four, five, six or more markers from Table 4. In certain instances, TAF renal MSCs can be more specifically identified by identifying a combination of markers that are more strongly expressed, such as 12-fold or more (optionally, a TPM threshold of >2000) than any combination of the aforementioned markers, such as HAVCR1 and / or CD24 and / or CLDN6 and / or ABCB1 and / or SHISA9 and / or CRB3, compared to TAF-MSCs. When a combination of markers is used, identification can be achieved using a lower threshold of stronger expression, such as 4-fold or more, 6-fold or more, or 8-fold or more expression of each marker.

[0239] In contrast to the above surface markers that can be expressed more strongly on the surface of TAF renal MSCs (positive markers), in certain instances, the following surface markers: GREM1, PDGFRB, BGN, FAP, CXCL12, CCKAR, CD248 can be expressed more weakly on TAF renal cells compared to other cell types (negative markers), such as 1 / 8-fold or less expression of any combination of the aforementioned markers and other TAF-derived cells and / or TAF-MSC cells (optionally, a TPM threshold of >500). When a combination of negative markers is used, identification can be achieved using a lower threshold of weaker expression, such as 1 / 2-fold or less, 1 / 4-fold or less, or 1 / 6-fold or less expression of each of the markers.

[0240] Combinations of two or more of these negative markers can also be used to more specifically isolate TAF renal MSCs. In addition, those skilled in the art will also recognize that combinations comprising both negative and positive markers, such as at any of the above thresholds, can also effectively and more specifically isolate TAF renal MSCs.

[0241] TAF skin cell marker

[0242] Similar to the TAF lung and kidney MSC markers identified above, a number of target surface markers associated with skin TAF cells were identified. For example, a non-exclusive list of surface markers used to identify and isolate TAF skin cells is provided in Table 5 below. The expression of the TAF skin MSC markers identified in Table 5 on prioritized clones may be increased by at least 12 times compared to ordinary TAF-MSC clones (optionally, TPM threshold>2000). In addition, due to the limited number of different MSC subtypes in TAF, the selection of tissue-specific MSCs can be completed by first characterizing and then gradually counter-selecting / sorting the materials by considering the combined (multivariate) surface marker profiles of different tissue-specific MSCs. It will be understood by those skilled in the art that any such combination of these surface markers can be used to identify and isolate TAF skin cells from general TAF-derived cells and / or TAF-MSC cell populations. In some instances, the following non-exclusive list of surface markers may be expressed higher on the surface of TAF skin cells compared to other cell types, such as other TAF-derived cells and / or TAF-MSC cells:

[0243] 1.TNFSF18-TNF superfamily member 18;

[0244] 2. PCDH19- protocadherin 19;

[0245] 3. NCAM2-neural cell adhesion molecule 2;

[0246] 4.TNFSF4-TNF superfamily member 4;

[0247] 5.CD248-endosialin;

[0248] 6.DDR2-discoid domain receptor tyrosine kinase 2;

[0249] 7.HTR2B-5-hydroxytryptamine receptor 2B;

[0250] 8. PCDH18- protocadherin 18;

[0251] 9.SULF1-sulfatase 1;

[0252] 10.MME-membrane metalloendopeptidase;

[0253] 11.ADGRA2-adhesion G protein-coupled receptor A2;

[0254] 12.DCSTAMP - seven-transmembrane protein expressed by dendritic cells;

[0255] 13.PDGFRA-platelet-derived growth factor receptor alpha;

[0256] 14.UNC5B-unc-5 spindle protein receptor B;

[0257] 15.SCUBE3-protein containing signal peptide, CUB domain and EGF-like domain 3;

[0258] 16.CEMIP-cell migration-inducing hyaluronidase 1;

[0259] 17.BDKRB1-bradykinin receptor B1;

[0260] 18.FLT1-fms-related tyrosine kinase 1;

[0261] 19.BDKRB2-bradykinin receptor B2;

[0262] 20.FAP-fibroblast activation protein alpha;

[0263] 21. CASP1 - caspase 1; and / or

[0264] 22.SRPX2-sushi repeat-containing protein X-linked 2.

[0265] Table 5

[0266] As will be appreciated by those skilled in the art, suitable combinations of the markers listed in Table 5 can be used to isolate TAF skin MSCs from TAF-MSCs by selecting specific markers from Table 5 or a combination of two, three, four, five, six, or more markers from Table 5. In certain instances, TAF skin MSCs can be more specifically identified compared to TAF-MSCs by identifying a combination of markers that are more strongly expressed, for example, 12-fold or more (optionally, TPM>2000) than any combination of the aforementioned markers, such as TNFSF18 and / or PCDH19 and / or NCAM2 and / or TNFSF4 and / or CD248 and / or DDR2. When a combination of markers is used, identification can be achieved using a lower threshold of stronger expression, such as 4-fold or more, 6-fold or more, or 8-fold or more expression of each marker.

[0267] In contrast to the above surface markers that may be more strongly expressed on the surface of TAF skin cells (positive markers), in certain examples, the following surface markers: CD24, TNFSF10, ITGB4, ABCB1 may be expressed more weakly on TAF skin cells compared to other cell types (negative markers), such as 1 / 8-fold or less expressed (optionally, TPM threshold > 500) for any combination of the aforementioned markers and other TAF-derived cells and / or TAF-MSC cells. When using a combination of negative markers, identification can be achieved using a lower threshold of weaker expression, such as 1 / 2-fold or less, 1 / 4-fold or less, or 1 / 6-fold or less expression of each of the markers.

[0268] Combinations of two or more of these negative markers can also be used to more specifically isolate TAF skin MSCs. In addition, those skilled in the art will also recognize that combinations including both negative and positive markers, such as at any of the above thresholds, can also effectively and more specifically isolate TAF skin MSCs.

[0269] TAF neural cell marker

[0270] Similar to the TAF lung, kidney and skin MSC markers identified above, many target surface markers associated with TAF neural cells have been identified. For example, a non-exclusive list of surface markers for identifying and separating TAF neural cells is provided below. The expression of the TAF neural MSC surface markers identified in Table 6 on prioritized clones is at least increased by 3 times (optionally, TPM threshold>500) compared to common TAF-MSC clones. In addition, due to the limited number of different MSC subtypes in TAF, the selection of tissue-specific MSC can first be completed by characterization, and then the material is gradually reversed / sorted by considering the combination (multivariate) surface marker spectrum of different tissue-specific MSCs. It will be understood by those skilled in the art that any such combination of these surface markers can be used to identify and separate TAF neural cells from general TAF derived cells and / or TAF-MSC cell populations. In some instances, compared to other cell types, such as other TAF derived cells and / or TAF-MSC cells, the following non-exclusive list of surface markers can be expressed higher on the surface of TAF neural cells:

[0271] 1.HAVCR1-Hepatitis A virus cellular receptor 1;

[0272] 2. ACKR3 - atypical chemokine receptor 3;

[0273] 3.OSCAR-osteoclast-associated Ig-like receptor;

[0274] 4.C3-complement C3;

[0275] 5.SIRPB1-signal regulatory protein β1;

[0276] 6.SLC6A6-solute carrier family 6 member 6;

[0277] 7.CCKAR-cholecystokinin A receptor;

[0278] 8.TNFSF10-TNF superfamily member 10;

[0279] 9.CLSTN2-calcine syntenin 2;

[0280] 10.TENM2-teneurin transmembrane protein 2;

[0281] 11.SFRP1-secreted frizzled-related protein 1;

[0282] 12.PIK3IP1-phosphatidylinositol 3-kinase interacting protein 1;

[0283] 13.SCNN1D-sodium channel epithelial 1 delta subunit;

[0284] 14.CLDN11-tight junction protein 11;

[0285] 15. ALDH3B1 - aldehyde dehydrogenase family 3 member B1; and / or

[0286] 16.ITGB4 - integrin subunit β4.

[0287] Table 6

[0288] As will be appreciated by those skilled in the art, suitable combinations of the markers listed in Table 6 can be used to separate TAF neural MSCs from TAF-MSCs by selecting specific markers from Table 6 or selecting a combination of two, three, four, five, six or more markers from Table 6. In some instances, TAF neural MSCs can be more specifically identified by identifying a combination of markers that are more strongly expressed, such as 3-fold or more of any combination of the aforementioned markers, such as HAVCR1 and / or ACKR3 and / or OSCAR and / or C3 and / or SIRPB1 and / or SLC6A6, compared to TAF-MSCs. When a combination of markers is used, identification can be achieved using a lower or higher threshold of stronger expression, such as 2-fold or more, 6-fold or more, 8-fold or more, or 12-fold or more of each marker. In addition, those skilled in the art will also recognize that a combination of both negative and positive markers, such as at any of the above thresholds, can also be effective in more specifically isolating TAF neural MSCs.

[0289] All features disclosed in this specification (including any accompanying drawings, claims, abstract and figures), and / or all steps of any method or process so disclosed, may be combined in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive. The disclosure is not limited to the details of any foregoing examples. The disclosure extends to any novel feature or any novel combination of features disclosed in this specification (including any accompanying claims, abstract and figures), or to any novel step or any novel combination of steps of any method or process so disclosed.

[0290] Those skilled in the art will appreciate that, in some instances, the actual steps taken in the illustrated or disclosed process may be different from those steps shown in the accompanying drawings. Depending on the instance, some of the above steps may be deleted, or other steps may be added. For example, the actual steps or sequence of steps taken in the disclosed process may be different from those shown in the drawings. Depending on the instance, some of the above steps may be deleted, or other steps may be added. In addition, the features and attributes of the specific examples disclosed above may be combined in various ways to form additional examples, all of which fall within the scope of this disclosure.

[0291] Unless otherwise specifically stated, or understood otherwise in the context of use, conditional language, such as "can," "could," "might," or "may," is generally intended to express that certain instances include certain features, elements, or steps, while other instances do not. Thus, such conditional language is generally not intended to imply that one or more instances require a feature, element, or step in any way, or that one or more instances must include logic for determining, with or without user input or prompting, whether such feature, element, or step is included in or to be performed in any particular instance. The terms "comprising," "including," "having," and the like are synonymous and are used inclusively in an open-ended manner and do not exclude additional elements, features, actions, operations, and the like. Furthermore, the term "or" is used in its inclusive sense (rather than in its exclusive sense) so that when used, for example, to connect a list of elements, the term "or" means one, some, or all of the elements in the list. Similarly, the term "and / or" with respect to a list of two or more items encompasses all of the following interpretations of the term: any one item in the list, all items in the list, and any combination of items in the list. Furthermore, as used herein, the term "each" may refer to any subset of a group of elements to which the term "each" applies, in addition to its ordinary meaning. Additionally, when used in this application, the words "herein," "above," "below," and words of similar meaning refer to this application as a whole and not to any particular portions of this application.

[0292] Unless expressly stated otherwise, linking language such as the phrase "at least one of X, Y, and Z" should be understood in context as generally used to convey that an item, term, etc. can be X, Y, or Z. Thus, such linking language is not generally intended to imply that certain instances require the presence of at least one of X, at least one of Y, and at least one of Z.

[0293] As used herein, language of degree, such as the terms "approximately," "about," "substantially," and "substantially" as used herein, refers to a value, amount, or characteristic that is close to a specified value, amount, or characteristic and still performs a desired function or achieves a desired result. For example, the terms "approximately," "about," "substantially," and "substantially" may refer to an amount that is within less than 10%, less than 5%, less than 1%, less than 0.1%, and less than 0.01% of a specified amount. As another example, in certain instances, the terms "substantially parallel" and "substantially parallel" refer to a value, amount, or characteristic that deviates from perfect parallelism by less than or equal to 15 degrees, 10 degrees, 5 degrees, 3 degrees, 1 degree, or 0.1 degrees.

[0294] Various modifications to the embodiments described in this disclosure will be readily apparent to those skilled in the art, and the general principles defined herein may be applied to other embodiments without departing from the spirit or scope of the disclosure. Therefore, the disclosure is not intended to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and features disclosed herein. Certain examples of the disclosure are included in the set of claims listed below or presented in the future.

Claims

1. A method for obtaining full-term amniotic fluid cells from full-term amniotic fluid, comprising: Provide full-term amniotic fluid; removing particulate matter from the full-term amniotic fluid to obtain purified full-term amniotic fluid cells; performing adhesion selection on the purified full-term amniotic fluid cells to obtain full-term amniotic fluid adherent cells; passaging the term amniotic fluid adherent cells to obtain a cell population comprising the term amniotic fluid cells; and The term amniotic fluid cells are selected from the population as cells expressing at least a surface marker which is CD248, wherein the term amniotic fluid cells are term amniotic fluid lung mesenchymal stem cells.

2. The method of claim 1 , wherein selecting term amniotic fluid cells further comprises selecting the term amniotic fluid cells from the population as cells expressing at least one marker selected from the group consisting of: PCDH19, DDR1, MME, IFITM10, BGN, NOTCH3, SULF1, TNFSF18, BDKRB1, FLT1, PDGFRA, TNFSF4, UNC5B, FAP, CASP1, DDR2, PCDH18, LRRC38, and CRLF1.

3. The method of claim 1, wherein the selecting step comprises selecting term amniotic fluid cells that further express a surface marker selected from the group consisting of DDR1 and LRRC38.

4. The method of claim 1, wherein the selecting step comprises selecting term amniotic fluid cells that further express DDR1 and LRRC38.

5. The method of any one of the preceding claims, wherein selecting the term amniocytes comprises excluding cells expressing a marker selected from the group consisting of CD24, ITGB4, TNFSF10, GFRA1, CD74, FGFR4, HAVCR1, and OSCAR.

6. The method of any one of claims 1 to 4, wherein removing particulate matter comprises filtering and centrifuging the term amniotic fluid.

7. The method of any one of claims 1 to 4, wherein subjecting the purified term amniocytes to adhesion selection comprises adhering the purified term amniocytes to a surface coated with a vitronectin substrate.

8. The method according to any one of claims 1 to 4, wherein the selecting step is performed using fluorescence activated cell sorting (FACS).

9. The method according to any one of claims 1 to 4, wherein the selecting step is performed using antibodies against any one of the markers or surface markers.

10. The method of any one of claims 1 to 4, wherein the selecting step comprises selecting term amniotic fluid cells that express at least two markers, at least three markers, or at least four markers from the surface markers of claim 2.

11. The method according to any one of claims 1 to 4, wherein the selecting step comprises a plurality of sorting steps, each sorting step comprising directing the term amniotic fluid cells into a first output group or a second output group based on a set of markers expressed or not expressed by the respective term amniotic fluid cells.

12. The method of claim 11, wherein the selecting step comprises: a first sorting step, directing term amniotic fluid cells expressing at least the surface marker CD248 into a first output set, and In a second sorting step, term amniotic fluid cells from the first output group that express a second set of markers are directed into a second output group.

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