FORM 2
THE PATENTS ACT, 1970 (39 of 1970) & THE PATENTS RULES, 2003
COMPLETE SPECIFICATION (See section 10, rule 13)
1. Title of the invention: METHOD FOR PRODUCING MESENCHYMAL STEM CELLS
FOR THERAPEUTIC APPLICATIONS
2. Applicant(s)

NAME

NATIONALITY

ADDRESS

REGROW BIOSCIENCES PRIVATE LIMITED

Indian

Regrow Biosciences Private Limited Ground Floor, 22, Shah Industrial Estate, Nagargaon, Pune, Maharashtra 410401, India

3. Preamble to the description
COMPLETE SPECIFICATION
The following specification particularly describes the invention and the manner in which it
is to be performed.
1
PD050890IN-SC

FIELD OF INVENTION
[001] The present disclosure relates to the field of mesenchymal stem cells (MSCs)
and, particularly to, a method for producing MSCs. It further relates to compositions
5 and therapeutic formulations comprising the MSCs.
BACKGROUND OF THE INVENTION
[002] Several varieties of drugs are available for treatment of autoimmune diseases
like rheumatoid arthritis (RA), which includes steroid hormones, biological agents,
immunosuppressant, anti-rheumatic drugs, and anti-inflammatory drugs. However,
10 such approaches have been observed to have undesirable side effects. Reported side
effects of RA treatment includes immune deficiency, gastrointestinal tract disorders, hormonal disturbances, and complications in cardiovascular system. Additionally, RA patients in some instances become resistant to long-term treatments. Thus, the current therapeutic approaches for arthritis and similar
15 diseases need an effective alternative involving biological substitutes, which is
economical, long effective and having reduced or no side effects.
SUMMARY OF INVENTION
[003] In an aspect of the present disclosure, there is provided a method of producing
mesenchymal stem cells (MSCs) for therapeutic application, the method
20 comprising: a) culturing a heterogeneous population of MSCs in a culture medium
for a period in a range of 20 to 35 days; b) staining the MSCs to obtain a heterogeneous population of stained MSCs; c) adding droplet encapsulation media, comprising a density gradient solution to the heterogeneous population of stained MSCs, to obtain a population of microfluidic droplets, wherein each droplet
25 preferably comprises a single MSC; d) providing the population of microfluidic
droplets obtained from step (c) to a microfluidics device; and e) identifying and selecting a population of homogeneous MSCs having medium size in the range of 15 to 30 µm; wherein the population of MSCs obtained from step (e) are viable MSCs, expressing MSC specific markers selected from CD 73, CD 90, and
30 combinations thereof; and exhibiting increased expression of genes selected from a
group consisting of COL12A gene, IGFBP5 gene, THBS2 gene, GREM1 gene, CDH2 gene, and combinations thereof.
2

[004] In an aspect of the present disclosure, there is provided a composition
comprising the MSCs obtained by the method as disclosed herein; and an excipient;
wherein the excipient is selected from DMEM, human serum albumin (HSA),
dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), or combinations thereof.
5 [005] In an aspect of the present disclosure, there is provided a formulation
comprising the MSCs obtained by the method as disclosed herein, or the composition as disclosed herein, and a pharmaceutically acceptable carrier. [006] In an aspect of the present disclosure, there is provided a method of treating a disease in a subject, comprising administering the MSCs obtained by the method
10 as disclosed herein or the formulation as disclosed herein to a subject.
[007] These and other features, aspects, and advantages of the present subject matter will be better understood with reference to the following description and appended claims. This summary is provided to introduce a selection of concepts in a simplified form. This summary is not intended to identify key features or essential
15 features of the claimed subject matter, nor is it intended to be used to limit the scope
of the claimed subject matter.
BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS
[008] The following drawings form a part of the present specification and are
included to further illustrate aspects of the present disclosure. The disclosure may
20 be better understood by reference to the drawings in combination with the detailed
description of the specific embodiments presented herein.
[009] Figure 1 depicts the Wharton’s jelly isolation from Umbilical Cord Tissue
(UCT): A. UCT (5-7 cm) piece received at manufacturing unit after the complete
discharge; B. The cross section of Umbilical Cord tissue, in accordance with the
25 embodiments herein.
[0010] Figure 2 is a schematic representation of the method for producing MSCs,
in accordance with the embodiments herein.
[0011] Figure 3 depicts A. Bright field image of single cell encapsulated in a
droplet; B. Representative image of an encapsulated cell labelling in deep learning
30 model; and C. Size distribution of MSCs and the Mean fluorescence Intensity
measurement of different sized MSCs, in accordance with the embodiments herein.
3

[0012] Figure 4 depicts the pie chart and bar chart indicating the unique genes
present in small and medium sized population, in accordance with the embodiments
herein.
[0013] Figure 5 is a representation of gene network of selected 64 genes that were
5 highly expressed in medium sized population when compared to small or large
sized population, in accordance with the embodiments herein.
DETAILED DESCRIPTION OF THE INVENTION
[0014] Those skilled in the art will be aware that the present disclosure is subject
to variations and modifications other than those specifically described. It is to be
10 understood that the present disclosure includes all such variations and
modifications. The disclosure also includes all such steps, features, compositions, and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any or more of such steps or features. Definitions
15 [0015] For convenience, before further description of the present disclosure, certain
terms employed in the specification, and examples are delineated here. These definitions should be read in the light of the remainder of the disclosure and understood as by a person of skill in the art. The terms used herein have the meanings recognized and known to those of skill in the art, however, for
20 convenience and completeness, particular terms and their meanings are set forth
below.
[0016] The articles “a”, “an” and “the” are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. [0017] The terms “comprise” and “comprising” are used in the inclusive, open
25 sense, meaning that additional elements may be included. It is not intended to be
construed as “consists of only”.
[0018] Throughout this specification, unless the context requires otherwise the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated element or step or group of elements
30 or steps but not the exclusion of any other element or step or group of elements or
steps.
4

[0019] The term “including” is used to mean “including but not limited to”.
“Including” and “including but not limited to” are used interchangeably.
[0020] Embodiments herein include a method for producing MSCs. MSCs are
increasingly being used as off the shelf products for treating various diseases.
5 Accordingly, embodiments herein achieve a method for producing MSCs for
therapy or therapeutic applications. The method, as disclosed herein, can achieve the production and expansion of medium sized MSCs having therapeutic potential. Further, the embodiments herein also provide a composition comprising the MSCs and an excipient. Embodiments herein also provide a formulation comprising the
10 MSCs and a pharmaceutically acceptable carrier. Accordingly, embodiments herein
also include a method for treating a disease in the subject using the composition of MSCs or the formulation.
[0021] Embodiments herein provide a method for producing MSCs. The term “mesenchymal stem cell” or “MSCs”, as used herein, refers to a cell or cell
15 population of multipotent cells. The MSCs, according to embodiments herein, are
characterized by the expression of one or more cell surface markers selected from CD73, CD90, or CD105. In further embodiments herein, the MSCs may be characterized by not expressing one or more of the markers selected from HLADR, CD34, or CD45.
20 [0022] The MSCs may be obtained from commercial sources or, alternatively,
derived from Umbilical Cord Tissue (UCT) of mammals, in particular humans. The MSCs, in accordance with the embodiments herein, are derived from the Wharton’s jelly in UCT of single or multiple donors. [0023] In an embodiment of the present disclosure, the heterogeneous population
25 of MSCs is obtained from human umbilical cord tissue (UCT) of single donor or
multiple donors.
[0024] In some embodiments, the donor for obtaining UCT are selected based on a set of physiological and medical history parameters. In an embodiment, the UCT donor is a healthy pregnant female with a negative association with the medical
30 history parameters selected from drug addiction; alcohol addiction; chronic heart
and respiratory diseases; malignancies; organ, tissue, or any past stem cell
5

transplant; past history of diabetes; joint arthroplasty; viral/vector borne diseases,
such as dengue, CJD, etc; hospitalization or surgery in the past year; cupping or
tattooing in the past year; dentistry in the past week; bypass/ angiography or
haemodialysis; high-risk sexual behaviors; domestic or international travel history
5 to a high-risk area in terms of infectious disease; bleeding problems; blood pressure
abnormalities; sepsis and fever of unknown origin; liver disease; autoimmune disease; hepatitis with unknown cause; genital ulcerative disease; disseminated lymphadenopathy and splenomegaly; bruising or skin lesions; neurological disorders; metabolic disorders or genetic disorders. In further embodiments, the
10 selected donor is vaccinated as per national vaccination schedule.
[0025] In some embodiments, the UCT donor is a healthy pregnant female with an acceptable criterion for physiological parameters identified through blood, serum, and urine analysis. The various physiological parameters and the corresponding acceptance criterion for selecting a donor for MSCs isolation is listed in Table A
15 below.
Table A

Blood, serological and urine analyses
Test Parameter Accepted criteria
1 HLA typing Must be done
2 Fasting plasma glucose test (FPG) 60-100 mg/dl
3 CRP Negative
4 Hb No Hb variant detected
5 Electrolytes
Na
K
Ca 135-145 mEq/L 3.5-5.3 mEq/L 8.3-1.3 mg/dl
6 Urea 11-50 mg/dl
7 Creatinine 0.5-1.4 mg/dl
8 Albumin 3.5-5.2 g/dl
9 Total bilirubin (direct and indirect) 0.3-1.2 mg/dl
10 Urinalysis Normal
11 Alkaline phosphatase 64-306 U/L
12 PT 12-14.5 seconds
13 PTT 22-37 seconds
14 SGOT <45 U/L
15 SGPT <45 U/L
6

16 LDH 100-190 U/L
17 CBC with differential Normal
18 HIV ½ Negative
19 HTLV ½

20 HBV

21 HCV

22 CMV

23 EBV

24 RPR Negative
25 Serum beta-HCG pregnancy <5 mIU/Ml
Pregnancy relevant test
1 Multiple marker test Negative
2 Ultrasound scan Normal
3 Alpha fetoprotein screening Normal
4 Delivery Full term (37-42 weeks)
5 Surrogacy case NA
[0026] In certain embodiments, the MSCs are isolated from UCT. Various methods are known in the art for the isolation of MSCs from UCT.
[0027] In an embodiment, the UCT are minced and subjected to enzymatic
5 digestion. In an embodiment, the heterogeneous population of MSCs is obtained by
treating the human umbilical cord tissue with at least one enzyme selected from collagenase, hyaluronidase, proteins identified from CB+MB (Cord Blood and Maternal Blood) plasma lysate [Peroxiredoxin 1 (PRDX1) and Heat Shock Protein -70 (HSP70)], or mixtures thereof. In another embodiment, the heterogeneous
10 population of MSCs is obtained by treating the human umbilical cord tissue with
collagenase, hyaluronidase, and proteins identified from CB+MB plasma lysate (PRDX1 and HSP70). Collagenase and hyaluronidase may be procured commercially from Thermofisher, Catalogue Number: 17100017 and Sigma-Aldrich, Catalogue Number: H3506, respectively. Various methods well known in
15 the art can be used to obtain the proteins, such as Peroxiredoxin 1 (PRDX1) and
Heat Shock Protein -70 (HSP70 from cord blood and maternal blood plasma. For example, the plasma proteins may be precipitated from the mixture of cord blood and maternal blood obtained from the same donor using protein precipitating agents, such as ammonium acetate, ethanol, or combination thereof.
20 [0028] The MSCs isolated from UCT may be a heterogeneous population of MSCs.
The term “heterogeneous population”, as used herein, refers to a mixed population
7

of small, medium, or large sized MSCs. According to embodiments herein, the
MSCs having medium size are particularly expanded and produced using the
method as described herein. The term “expanded” or “expanded population”, as
used herein, refers to an increased population of cells, wherein the number of cells
5 is higher as compared to before expansion. It refers to a multiplied cell population.
[0029] In an embodiment, there is provided a method of producing mesenchymal stem cells (MSCs) for therapeutic application, the method comprising: a) culturing a heterogeneous population of MSCs in a culture medium for a period in a range of 20 to 35 days; b) staining the MSCs to obtain a heterogeneous population of stained
10 MSCs; c) adding droplet encapsulation media, comprising a density gradient
solution to the heterogeneous population of stained MSCs, to obtain a population of microfluidic droplets, wherein each droplet preferably comprises a single MSC; d) providing the population of microfluidic droplets obtained from step (c) to a microfluidics device; and e) identifying and selecting a homogeneous population of
15 MSCs of medium size in the range of 15 to 30 µm; wherein the population of MSCs
obtained from step (e) are viable MSCs, expressing MSC specific markers selected from CD 73, CD 90, and combinations thereof; and exhibiting increased expression of genes selected from a group consisting of COL12A gene, IGFBP5 gene, THBS2 gene, GREM1 gene, CDH2 gene, and combinations thereof.
20 [0030] Culturing, according to embodiments herein, refers to growing or
maintaining MSCs by providing the physical conditions (for eg: temperature) and chemical conditions (for eg: buffers, growth factors, nutrients, vitamins, etc). [0031] The medium for culturing MSCs, according to the present disclosure, is a medium capable of proliferating MSCs. Various MSCs culture media are known
25 and may be used in embodiments herein. Examples of MSC culture media include,
but are not limited to, Dulbecco’s Modified Eagle Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM), DMEM-F12, F12, Minimum Essential Medium α (ALPHA-MEM), etc. In an embodiment, the culture medium is selected from DMEM, IMDM, DMEM-F12, F12, ALPHA-MEM, or combinations thereof.
30 In another embodiment, the medium is Dulbecco’s Modified Eagle Medium
(DMEM) (Thermo Fisher) with 10% of Fetal Bovine Serum (FBS) (EDQM
8

Certified), BMP4 and 1ng to 10 ng per ml of Fibroblast Growth Factor (FGF) (Thermofisher).
[0032] The MSCs culture medium may be replaced every 2-7 days. In an
embodiment, the MSCs culture medium is replaced every 2 to 7 days. In another
5 embodiment, the MSCs culture medium is replaced every 2 to 3 days, 2 to 4 days,
2 to 5 days, 2 to 6 days, or 2 to 7 days.
[0033] In some embodiments, the heterogeneous MSC population is cultured to a confluence of at least 80%, but less than 100%, more preferably the heterogeneous MSC population is cultured to a confluence of 80% to 90%.
10 [0034] Upon achieving the desired confluency, the MSCs may be trypsinized to
detach the suspended cells from the culture flasks and reseeded for adherence. The culturing and optional trypsinization can be repeated so that the MSC-containing population of cells achieves a desired passage number. Typically, the initial population of cells P0 (passage 0) is the cells that have been derived from UCT.
15 When P0 is defined as the extracted population, then the MSCs expanded up to P3
or P4 is sufficient to obtain enough medium-sized MSCs.
[0035] According to embodiments herein, passage 3 (P3) is adequate to produce a MSC population with therapeutic potential. [0036] In an embodiment of the present disclosure, the step (a) further comprises
20 reseeding the heterogeneous population of MSCs at a cell density in the range of
5000 to 11,000/cm2 and expanding the heterogeneous population of MSCs up to passage 3.
[0037] After every passage, the MSCs may be monitored for its attachment status, cell morphology, cell growth and microbial contamination, if any. The remaining
25 MSCs may be cryopreserved using suitable cryostoring solutions, for eg:
Cryostor™ containing 10% DMSO. In an embodiment, cryopreservation is performed in cryotubes containing 10% DMSO, at a temperature in the range -75 to -85°C, preferably -80°C temperature or in liquid nitrogen tanks. The cryopreserved MSCs can be thawed later for further expansion.
30 [0038] In an embodiment, the heterogeneous population of MSCs in step (b) are
stained using a cell viability stain or MSC marker stain.
9

[0039] Various methods are known in the art to assess the viability status of culture
expanded MSCs. For example, MSCs are stained using certain cell viability stains
that help in differentiating the viable MSCs and non-viable MSCs. Similarly,
various MSC marker stains are known in the art to determine the characteristic
5 expression of MSC cell surface markers, and to confirm the identity of MSCs. In
an embodiment, the cell viability stain is selected from carboxyfluorescein diacetate succinimidyl ester (CFSE), trypan blue, propidium iodide (PI), or combinations thereof; and the MSC marker stain is selected from CD73, CD90, CD105, or combinations thereof; and wherein the heterogeneous population of MSCs is having
10 a concentration in the range of 0.5 to 5x106 cells/ml, preferably having a
concentration of 1x106 cells/ml.
[0040] The MSCs of P3 are validated for their viability and MSC marker status and may be further subjected to sorting or for particularly separating the medium sized MSCs from the culture expanded heterogeneous population of MSCs. MSCs can
15 be fractioned to small, medium, or large-sized MSC populations by any modality
suitable to isolate these populations from an MSC-containing population, such as a heterogeneous MSC population. Exemplary modalities for separation of medium-sized MSCs include microfluidic devices, size exclusion filters, and density columns. According to embodiments herein, the heterogeneous MSCs are
20 processed in a microfluidics platform to separate out the medium-sized MSCs.
Examples of microfluidics platform that are well known in the art, that could be used are droplet, capillary, pressure, acoustics, or electrical based microfluidics platforms. In an embodiment, the microfluidics device is droplet microfluidic platform.
25 [0041] In an embodiment, the heterogeneous population of stained MSCs are
encapsulated in a droplet, by a droplet generation device. According to embodiments herein, a droplet encapsulation media is added to the heterogeneous population of stained MSCs to obtain a population of microfluidic droplets, wherein each droplet preferably comprises a single MSC.
30 [0042] In an embodiment, the droplet encapsulation media comprises a density
gradient solution, wherein the density gradient solution is selected from an
10

iodixanol density gradient solution or a sucrose gradient solution. In another
embodiment, the droplet encapsulation media comprises a density gradient solution,
wherein the density gradient solution is an iodixanol density gradient solution
(Opti-prep density gradient solution). The droplet encapsulation media further
5 comprises a MSCs culture medium, such as DMEM as described hereinabove to
maintain the viability of MSCs during droplet generation. Examples of droplet generation device that are known in the art that could be used are active droplet formation (electric, magnetic, centrifugal) and passive droplet formation (cross-flowing, flow focusing, co-flowing) devices.
10 [0043] In an embodiment, the size of the microfluidic droplet is in the range of 100
to 200 micron; and each of the microfluidic droplet is sufficient to allow the MSCs to replicate 3 to 4 times. In another embodiment, the size of the microfluidic droplet is in the range of 120 to 150 micron. [0044] In an embodiment, the step of the disclosed process further comprises
15 imaging the MSCs in the population of microfluidic droplets to record the size of
the MSCs; and subjecting the population of microfluidic droplets to automated size-based sorting to obtain a population of homogeneous MSCs having medium size. [0045] The microfluidic droplets are sorted or analysed in a microfluidic device and imaged using microscopy. Various microscopy techniques that can be used to
20 image the droplets are fluorescence microscopy, confocal microscopy, optical
microscopy, electron microscopy etc., In an embodiment, the microscopy technique used for imaging MSCs is bright field fluorescence microscopy.
[0046] In an embodiment, the microfluidic droplets are analysed at a flow rate in the range of 100 to 200 nanolitre/sec, preferably at a flow rate of 150 nanolitre/sec.
25 [0047] In certain embodiments, the stained heterogeneous population of MSCs are
sorted based on the cell size of the encapsulated MSCs, their cell viability and MSC marker stain status. The sorted droplets are lodged into nanowells. In an embodiment, 100 microfluidic droplets are lodged into nanowells of the microfluidic device.
30 [0048] In an embodiment, the population of microfluidic droplets are subjected to
automated size-based sorting to obtain a homogeneous population of MSCs having
11

medium size. The automated size-based sorting may be performed using various
methods known in the art, such as using artificial intelligence (AI)-based tool.
[0049] In an embodiment, the medium-sized MSCs are sorted from the population
of microfluidic droplets using automated size-based sorting, preferably using an
5 artificial intelligence-based tool trained for size-based identification of cells.
[0050] According to the present disclosure, the artificial intelligence-based tool for automated size-based sorting of MSCs is developed using python algorithms to train the model in size-based sorting of small, medium- and large sized MSCs. Alternatively, Fluorescence Activated Cell Sorting using flow cytometry could also
10 be used for size-based sorting of MSCs.
[0051] The term “medium-sized MSCs” as used herein refers to a population of MSCs characterized by the size in the range of 15 to 30 µm. The term “small-sized MSCs” as used herein refers to a population of MSCs characterized by having a size less than 15 µm, preferably 12.2 ±1.9 µm. The term “large-sized MSCs” as
15 used herein refers to a population of MSCs characterized by having a size greater
than 30 µm, preferably, 35.8 ± 6.7 µm.
[0052] In an embodiment, the population of MSCs of medium size is characterized by having a size in the range of 15 to 30 µm. In another embodiment, the population of MSCs of medium size is characterized by having a size in the range of 17 to 22
20 µm.
[0053] In an embodiment, the homogeneous population of MSCs comprises at least 50%, at least 58% to 60%, at least 70%, or at least 80% of medium-sized MSCs. [0054] In further embodiments, the medium-sized MSCs are characterized by the expression of certain genes that aid their therapeutic potential. In an embodiment,
25 the medium-sized MSCs are characterized by the increased expression of genes
selected from COL12A, IGFBP5, THBS2, GREM1, CDH2, CLDN11, POSTN, COL11A1, CDH11, CCL2, SLC7A2, COL8A1, GJA1, CCDC80, ACTG2, ANTXR1, THSD4, RND3, MXRA5, NTM, TEAD1, YAP1, CSPG4, RP11-166D19.1, TPBG, FAP, LAMC2, LUM, NUAK1, RAB3B, GPX8, DSC3,
30 LRRC17, GPC6, FBLN1, ADAMTS1, NR2F2, SEMA5A., SLIT2, TBX3, LARP6,
HAS2, FAM101A, FLRT2, CD276, NUPR1, TJP1, PTPN13, EVC, KCNG1,
12

PAMR1, PDLIM3, STC1, SCARA3, PRRX1, GPC1, SSTR1, DDX3Y, MOXD1,
RGS4, TBX2, CPA4, IGFBP6, VAT1L, or combination thereof. In another
embodiment, medium-sized MSCs are characterized by the increased expression of
genes selected from a group consisting of COL12A gene, IGFBP5 gene, THBS2
5 gene, GREM1 gene, CDH2 gene, and combinations thereof.
[0055] In an embodiment, the method further comprises, expanding the medium size MSCs up to passage 6 or up to passage 20 in culture; and optionally cryopreserving the medium size MSCs. The cryopreserved MSCs are also referred to herein as “cell bank”. The cell bank of medium-sized MSCs can be thawed to be
10 used for therapy or in therapeutic applications as such or in the form of a
composition or formulation according to various embodiments herein. [0056] Embodiments herein provide a composition comprising the medium-sized MSCs obtained by the method described in the present disclosure. [0057] In an embodiment, there is provided a composition comprising the MSCs
15 obtained by the method as disclosed herein; and an excipient; wherein the excipient
is selected from DMEM, human serum albumin (HSA), dimethyl sulfoxide (DMSO), fetal bovine serum (FBS), cord blood and maternal blood plasma or combinations thereof. [0058] Embodiments herein provide a formulation comprising the medium-sized
20 MSCs obtained by the method described in the present disclosure, or the
composition as disclosed herein.
[0059] In an embodiment, there is provided a formulation comprising the MSCs obtained from the method as described herein, or the composition as disclosed herein, and a pharmaceutically acceptable carrier.
25 [0060] The term “pharmaceutically acceptable carrier” as used herein refers to any
carrier including adjuvants, solvents, etc. that are suitable for preparing therapeutic
stem cell formulations. Various carriers are known to a person skilled in the art and
may be used in various embodiments herein. In an embodiment, the
pharmaceutically acceptable carrier is selected from ringer lactate solution, saline,
30 dextrose, heparin, or combinations thereof.
13

[0061] Embodiments herein further include a method of treating a disease in a subject.
[0062] The term “subject”, as used herein, refers to mammals, e.g., human and non-
human mammals. Examples of non-human animals include non-human primates,
5 dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, mice, rats, hamsters, guinea
pigs and etc. Unless otherwise noted, the terms “patient” or “subject” are used herein interchangeably. Preferably, the subject is human.
[0063] In an embodiment, there is provided a method of treating a disease in a subject, comprising administering the MSCs obtained from the method as described
10 herein or the formulation as disclosed herein to a subject.
[0064] In an embodiment, the MSCs are allogenic to the subject.
[0065] In an embodiment, the disease is selected from a group consisting of autoimmune diseases, autoimmune diseases (Rheumatoid Arthritis (RA), Acquired Aplastic Anemia, Acquired Hemophilia, Agammaglobulinemia, Alopecia Areata,
15 Ankylosing Spondylitis (AS), Anti-NMDA Receptor Encephalitis,
Antiphospholipid Syndrome (APS), Arteriosclerosis, Autoimmune Addison’s Disease (AAD), Autoimmune Autonomic Ganglionopathy (AAG), Autoimmune Encephalitis (AE)/ Acute Disseminated Encephalomyelitis (ADEM), Autoimmune Gastritis, Autoimmune Hemolytic Anemia (AIHA), Autoimmune Hepatitis,
20 Autoimmune Hyperlipidemia, Autoimmune Hypophysitis/Lymphocytic
Hypophysitis, Autoimmune Inner Ear Disease (AIED), Autoimmune Lymphoproliferative Syndrome (ALPS), Autoimmune Myelofibrosis (AIMF), Autoimmune Myocarditis, Autoimmune Oophoritis, Autoimmune Pancreatitis (AIP), Autoimmune Polyglandular Syndromes (APS), Autoimmune Progesterone
25 Dermatitis (APD), Autoimmune Retinopathy (AIR), Autoimmune Sudden
Sensorineural Hearing Loss, Balo Disease/Concentric Sclerosis, Behçet’s Disease, Birdshot Chorioretinopathy/Birdshot Uveitis, Bullous Pemphigoid, Castleman Disease, Celiac Disease, Chagas Disease, Chronic Inflammatory Demyelinating Polyneuropathy (CIDP), Chronic Autoimmune Urticaria, Churg-Strauss
30 Syndrome/Eosinophilic Granulomatosis with Polyangiitis (EGPA), Cogan's
Syndrome (CS), Cold Agglutinin Disease (CAD), Crest Syndrome, Crohn's
14

Disease, Stricturing Crohn's Disease, Cronkhite-Canada Syndrome (CCS),
Cryptogenic Organizing Pneumonia (COP), Dermatitis Herpetiformis (DH),
Dermatomyositis, Diabetes, Type 1 (T1D), Discoid Lupus Erythematosus (DLE),
Dressler’s Syndrome/ Post myocardial Infarction/ Post pericardiotomy Syndrome,
5 Eczema/Atopic Dermatitis, Eosinophilic Fasciitis, Erythema Nodosum, Essential
Mixed Cryoglobulinemia, Evans Syndrome, Fibrosing Alveolitis/Idiopathic Pulmonary Fibrosis (IPF), Giant Cell Arteritis/Temporal Arteritis/Horton's Disease, Giant Cell Myocarditis, Glomerulonephritis (GN), Goodpasture’s Syndrome/Anti-Gbm/Anti-Tbm Disease, Granulomatosis With Polyangiitis (GPA)/Wegener's
10 Granulomatosis, Graves’ Disease (GD), Guillain-Barrè Syndrome (GBS),
Hashimoto’s Thyroiditis/Autoimmune Thyroiditis, Henoch-Schölein Purpura (HSP)/Iga Vasculitis, Hidradenitis Suppurativa, Hurst’s Disease/Acute Hemorrhagic Leukoencephalitis (AHLE), Hypogammaglobulinemia, Iga Nephropathy/Berger’s Disease, Immune-Mediated Necrotizing Myopathy
15 (IMNM), Immune Thrombocytopenia (Itp)/Autoimmune Thrombocytopenia
Purpura, Inclusion Body Myositis (IBM), Igg4-Related Sclerosing Disease (ISD), Interstitial Cystitis, Juvenile Idiopathic Arthritis (Jia)/Adult-Onset Still's Disease, Juvenile polymyositis/Juvenile dermatomyositis/ juvenile myositis, Kawasaki disease, Lambert-Eaton Myasthenic Syndrome (LEMS), Leukocytoclastic
20 vasculitis, Lichen Planus, Lichen Sclerosus, Ligneous conjunctivitis, Linear Iga
Disease (LAD), Lupus Nephritis (LN), Lyme Disease/Chronic Lyme Disease/Post-Treatment Lyme Disease Syndrome (PTLDS), Lymphocytic colitis/microscopic colitis, Lymphocytic hypophystitis/autoimmune hypophystitis, Ménière’s Disease, Microscopic Polyangiitis (MPA)/ANCA-Associated Vasculitis, Mixed Connective
25 Tissue Disease (MCTD), Mooren’s ulcer, Mucha-Habermann disease, Multifocal
motor neuropathy, Multiple Sclerosis (MS), Myalgic Encephalomyelitis/Chronic
Fatigue Syndrome (ME/CFS), Myasthenia Gravis (MG), Narcolepsy,
Neuromyelitis Optica/Devic's Disease, Ocular Cicatricial Pemphigoid,
Opsoclonus-myoclonus syndrome (OMS), Palindromic Rheumatism,
30 Paraneoplastic Cerebellar Degeneration (PCD), Paraneoplastic Pemphigus, Parry-
Romberg Syndromeherth (PRS)/Hemifacial Atrophy (HFA)/Progressive Facial
15

Hemiatrophy, Paroxysmal Nocturnal Hemoglobinuria (PNH), Peripheral
uveitis/pars planitis, PANS/PANDAS, Parsonage-Turner Syndrome (PTS),
Pemphigoid Gestationis (PG), Pemphigus Foliaceus, Pemphigus Vulgaris,
Pernicious anemia, POEMS Syndrome, Polyarteritis Nodosa (PAN), Polymyalgia
5 Rheumatica, Polymyositis, Postural Orthostatic Tachycardia Syndrome (Pots),
Primary Biliary Cirrhosis (PBC), Primary Sclerosing Cholangitis (PSC), Psoriasis, Palmoplantar Pustulosis (PPP), Psoriatic Arthritis, Pulmonary fibrosis, idiopathic (IPF), Pure Red Cell Aplasia (PRCA), Pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud’s Syndrome, Reactive Arthritis, Reflex sympathetic
10 dystrophy syndrome (RSD)/Complex regional pain syndrome (CRPS), Relapsing
Polychondritis (RP), Restless leg syndrome (RLS)/ Willis-Ekbom disease, Rheumatic Fever, Sarcoidosis, Schmidt Syndrome/Autoimmune Polyendocrine Syndrome Type II, Scleritis, Scleroderma, Sclerosing Mesenteritis/Mesenteric Panniculitis, Serpiginous choroidopathy, Sjögren's Syndrome, Stiff person
15 syndrome (SPS), Small Fiber Sensory Neuropathy (SFSN), Small Fiber Sensory
Neuropathy (SFSN), Systemic Lupus Erythematosus (SLE), Subacute bacterial
endocarditis (SBE), Subacute cutaneous lupus, Susac’s syndrome, Sydenham’s
Chorea, Sympathetic ophthalmia, Takayasu’s arteritis (vasculitis), Testicular
Autoimmunity, Tolosa-Hunt syndrome, Transverse myelitis (TM),
20 Tubulointerstitial nephritis uveitis syndrome (TINU), Ulcerative Colitis,
Undifferentiated Connective Tissue Disease, Uveitis, Vasculitis, VEXAS Syndrome, Vogt-Koyanagi-Harada syndrome (VKH), Osteoarthritis, AVN, vertebral compression factor, urethral stricture, Sjogren’s syndrome and ureteric stricture), arthritis, Type I Diabetes, multiple sclerosis, inflammatory bowel
25 diseases and acromegaly.
[0066] Although the subject matter has been described with reference to specific embodiments, this description is not meant to be construed in a limiting sense. Various modifications of the disclosed embodiments, as well as alternate embodiments of the subject matter, will become apparent to persons skilled in the
30 art upon reference to the description of the subject matter. It is therefore
16

contemplated that such modifications can be made without departing from the spirit or scope of the present subject matter as defined.
EXAMPLES
[0067] The disclosure will now be illustrated with working examples, which is
5 intended to illustrate the working of disclosure and not intended to take restrictively
to imply any limitations on the scope of the present disclosure. Unless defined
otherwise, all technical and scientific terms used herein have the same meaning as
commonly understood to one of ordinary skill in the art to which this disclosure
belongs. Although methods and materials similar or equivalent to those described
10 herein can be used in the practice of the disclosed methods and compositions, the
exemplary methods, devices, and materials are described herein. It is to be understood that this disclosure is not limited to particular methods, and experimental conditions described, as such methods and conditions may vary.
Example 1: Isolation of MSCs from umbilical cord tissue (UCT)
15 1. UCT Donor selection and Evaluation
[0068] The umbilical cord tissue (UCT) sample was collected from a healthy volunteer (pregnant females) after receipt of written informed consent, which was obtained according to ISSCR (ICMR) guidelines and approval of the Institutional Ethics Committee, Regrow Biosciences Pvt. Ltd. for the use of intended human
20 biological sample for manufacturing and therapeutic goals (Reg.No:
ECR/309/Inst/MH/2019/RR-22).
[0069] Eligible, healthy volunteers for UCT were carefully chosen according to the inclusion criteria and exclusion criteria approved by Institutional Ethics Committee, Regrow Biosciences Pvt. Ltd (Table 1). Briefly donor competency referred to
25 general health through asking volunteer’s medical history, physical examination,
serological testing to insure the medical health and absence of viral infections, chest X-ray and electrocardiogram. The health certificate from the physician was necessary to confirm the qualification of the eligible donor. Table 1: Medical eligibility and suitability of UCT females
Donor medical history
17

Sr. No. History and physical examination Accepted criteria
1 Drug addiction Negative
2 Alcohol addiction Negative
3 Chronic heart and respiratory diseases Negative
4 Malignancies Negative
5 Organ, tissue, or any post stem cell transplant Negative
6 Vaccination According to national vaccination schedule
7 General anaesthesia Negative
8 Hospitalization or surgery in the past year Negative
9 Cupping or tattooing in the past year Negative
10 Dentistry in the past week Negative
11 Bypass/ Angiography or haemodialysis Negative
12 High-risk sexual behaviors Negative
13 Domestic or international travel history to a high-risk area in terms of infectious disease Negative
14 Bleeding problems Negative
15 Blood pressure Negative
16 Sepsis and fever of unknown origin Negative
17 Liver disease Negative
18 Autoimmune diseases Negative
19 Hepatitis with unknown cause Negative
20 Genital ulcerative disease Negative
21 Disseminated lymphadenopathy and splenomegaly Negative
22 Bruising or skin lesions Negative
23 Neurological disorders/conditions Negative
24 Metabolic disorders Negative
25 Genetic disorders Negative
Blood, serological and urine analyses
Test Accepted criteria
1 HLA typing Must be done
2 Fasting plasma glucose test (FPG) 60-100 mg/dl
3 CRP Negative
4 Hb No Hb variant detected
5 Electrolytes
Na
K
Ca 135-145 mEq/L 3.5-5.3 mEq/L 8.3-1.3 mg/dl
6 Urea 11-50 mg/dl
18

7 Creatinine 0.5-1.4 mg/dl
8 Albumin 3.5-5.2 g/dl
9 Total bilirubin (direct and indirect) 0.3-1.2 mg/dl
10 Urinalysis Normal
11 Alkaline phosphatase 64-306 U/L
12 PT 12-14.5 seconds
13 PTT 22-37 seconds
14 SGOT <45 U/L
15 SGPT <45 U/L
16 LDH 100-190 U/L
17 CBC with differential Normal
18 HIV 1/2 Negative
19 HTLV 1/2

20 HBV

21 HCV

22 CMV

23 EBV

24 RPR Negative
25 Serum beta-HCG pregnancy <5 mIU/mL
Pregnancy relevant test
1 Multiple marker test Negative
2 Ultrasound scan Normal
3 Alpha fetoprotein screening Normal
4 Delivery Full term (37-42 weeks)
5 Surrogacy case NA
2. Umbilical Cord tissue collection, transportation, and suitability testing
[0070] Umbilical cord tissue samples under a sterile condition were taken from full-
term and healthy female new-borns by normal/caesarean section. The umbilical
cord tissue was transferred to the cell manufacturing unit, within 72 h in phosphate
5 buffered saline (PBS, HiMedia) at 15-28°C. Approximately, 10-15 cm length and
6 to 9 mm thick umbilical cord tissue was cut into two and transferred into a pre-
labelled sterile container containing 20 ml of (1X) DPBS buffer solution
supplemented with penicillin (100 U/ml), streptomycin (100 µg/ml) and
gentamycin (Gibco) (10 µg/ml). Maternal and Cord blood (2 ml each) were also
10 collected in EDTA vial for reconfirming the Transfusion transmissible diseases
such as infectious diseases such as HIV 1 & 2 antibodies, HCV antibodies, Anti-HBc, HBsAg, HTLV 1 & 2, Syphilis antibodies, Malarial parasite, CMV IgM and IgG (Table 2). Table 2: Transfusion transmissible disease screening and karyotyping
19

Transfusion transmissible diseases tests
Test CT-MSC-01 CT-MSC-02 CT-MSC-03
HIV 1&2 antibodies Negative Negative Negative
HCV antibodies Non Reactive Non Reactive Non Reactive
Anti-HBc Non Reactive Non Reactive Non Reactive
HBsAg Non Reactive Non Reactive Non Reactive
HTLV 1&2 Negative Negative Negative
Syphilis antibodies Non-Reactive Non-Reactive Non-Reactive
Malarial parasite Not Detected Not Detected Not Detected
CMV IgM / IgG Negative/Immune Negative/Immune Negative/Immune
Other tests
Karyotyping Normal Normal Normal
3. Isolation of MSCs from Umbilical Cord Tissue
[0071] After obtaining Umbilical Cord tissue, the blood cells, arteries, and vein
were removed from the umbilical cord tissue (Figure 1A & B) and the remaining
Wharton's jelly were minced into small pieces. Briefly, the Umbilical Cord tissue
5 was cut into 1-2 cm sized pieces after removal of the blood vessel and repeated
washing was done with (1X) Ddpbs
[0072] buffer solution supplemented with antibiotics 100 U/ml of penicillin (Invitrogen), 100μg/ml of streptomycin (Invitrogen) and 10µg/ml of gentamicin (Gibco) in a biosafety cabinet. Half of these pieces were incubated with 1-5 mg/ml
10 Collagenase type-1 (Gibco) and 2-3 mg/ml hyaluronidase (Sigma) and rest half
were incubated with 1-5 mg/ml Collagenase type-1 (Gibco) and 2-3 mg/ml hyaluronidase (Sigma) along with proteins identified from CB+MB plasma lysate (PRDX and HSP70) for 2-6 hours at 37 ºC on shaking incubator. After complete digestion of tissue, the samples containing enzyme solution (treated umbilical cord
15 matrix reaction solution) were filtered by using a 40-100 µm cell strainer, to obtain
a filtrate solution. The filtrate solution (filtered solution) was centrifuged at 1100-1800 rpm for 5-10 minutes. The supernatant were discarded, and the pellet were dissolved in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher) with 10% of Fetal Bovine Serum (FBS) (EDQM Certified), BMP4 and 1ng to 10ng per
20

ml of Fibroblast Growth Factor (FGF) (Thermofisher), preferably 5 ng/ml to obtain
a stem cell suspension. The stem cell suspension was seeded into cell culture flask
followed by incubation at 37 ºC in a humidified atmosphere with 5% CO2 to obtain
a culture flask-adhered cultured stem cells. The culture flask- adhered cultured stem
5 cells were allowed to expand until it reached about 70-80% confluence.
Morphology of isolated and expanded mesenchymal stem cells (MSCs) was assessed using an inverted microscope, and the results were recorded for cell count, cell viability and colony forming unit (CFU) (Table 3). It is evident that the use of a combination of enzymes, such as Collagenase and Hyaluronidase along with
10 proteins identified from CB+MB plasma lysate (PRDX1 and HSP70) in the
enzymatic digestion of UCT resulted in improved viability of the isolated MSCs. 4. Procedure for obtaining CFUs:
[0073] About 100 MSCs were plated per 100-mm tissue culture dish (Falcon) in a complete culture medium. Then the cells were incubated for 10–14 days at 37 °C
15 in 5% humidified CO2. The media was removed after 14 days, washed with PBS,
and stained with 0.5% crystal violet in methanol for 5–10 min at room temperature. Following which, the plates were washed with PBS twice and the MSCs colonies that were violet colored were observed under the microscope and the colonies were counted.
20 Table 3: Enzymatic isolation procedure and assessment of cell count, viability and
CFU F (Colony Forming Unit - Fibroblast).

S. No Test Combination Test Parameter

Cell Count (In10^6) Cell Viability (In %) MSC CFU (No of colonies)
Sample ID Sample ID Sample ID
CT- CT- CT- CT- CT- CT- CT- CT- CT-
MSC MSC MSC MSC MSC MSC MSC MSC MSC
-01 -02 -03 -01 -02 -03 -01 -02 -03
1 Enzymatic digestion using Collagenase and Hyaluronidase 4.2 4.0 4.3 94. 5 95. 1 94. 9 3 5 4
21

2 Enzymatic digestion using Collagenase and
Hyaluronidase along with proteins identified from CB+MB plasma lysate (PRDX1 and HSP70) 5.5 5.8 6 96. 2 97. 3 97. 1 7 6 8
Example 2: Production of medium sized MSCs
1. Expansion of hMSCs up to passage 3
[0074] The schematic process flow to produce the medium-sized MSCs is shown in
Figure 2. After isolation and adherence, the cultured cells were subjected to
5 intermittent medium changes at every 48-72h intervals. This process was repeated
up to 4-5 times depending on the growth of culture to achieve 70-80% confluency. Afterwards sub-culturing was carried out with the help of trypsin enzyme to enhance the growth or expansion of cells. For detachment of cells, PBS wash followed by trypsinization, and 2-3 min incubation was carried out by using 0.25%
10 trypsin (GIBCO, Cat no. 25200056). Once the cells were found to be detached,
immediately 0.5 ml fetal bovine serum was added to stop the enzymatic activity and cells were centrifuged at 1300 rpm for 5 min. The supernatant media and cell samples were taken for mycoplasma and sterility quality control (QC) testing. The cell viability, cell count, and cell characterization were performed to check the
15 quality of cells. The cells were reseeded in T75 flask (cell density: about 5000-
11,000/cm2) for further expansion and were monitored for its attachment status, cell morphology, cell growth and microbial contamination, if any. The remaining cells were cryopreserved for cells for future use. The similar procedure was followed up to passage 3 (P3) expansion and cryopreservation.
20 2. Cell quality and characterization
[0075] At the level of P3, cells were characterized and evaluated with their identification markers and cultured conditions to confirm the healthy state of cells (Table 4).
22

[0076] Cells were examined with the routine QC testing protocols to confirm the pure population.
Table 4: Characterization of cells and quality assessment of isolated and expanded population at P3.

S.No. Test Specification Test Results
1 Cell number Over 1000,000 cells/vial 1000,000 cells/vial
2 Cell viability ≥80% Dye-Excluding cells 99%
3 Microbial Sterility Negative/no growth Negative/no growth
4 Endotoxin < 3 EU/ml < 0.750 EU/ml
5 Mycoplasma Negative Negative
6 Non -viable purities <1g/dL 0.375g/dL
7 Karyotyping Metaphase stage of mitosis 46, XX or 46, XY i.e Normal female/male karyotype pattern
8 Immunophenotyping >80% (For positive markers) 99.696% CD90 99.223% CD73 98.050% CD105

<10 % (For negative markers) 0.408% HLADR 2.893% CD34 3.317% CD45
5 3.Microfluidic characterization
Single cell droplet encapsulation and identification of characteristic population
[0077] At the stage of P3, cells (heterogeneous population of MSCs) were processed
for Microfluidics analysis. For droplet encapsulation the single cell suspension
(1x106 cells/ml) was made and stained with desired markers. The staining
10 procedure followed is briefed below:
1. 1x106 cells/ml media was taken in a 1.5 ml Eppendorf tube.
2. The cells were centrifuged at 180g for 5 min at 4ºC.
3. The media was discarded without disturbing the pellet and the cells were
resuspended in fresh 1ml PBS.
15 4. The cells were mixed with gentle pipetting so that single cell suspension would
be made. 5. 500 µl of suspension solution were centrifuged and washed with dPBS and centrifuged at 180g for 5 min at 4 ºC temperature.
23

6. The pellet was resuspended into 1ml PBS.
7. The 1ml solution was divided into 2 parts- 400 µl and 600 µl.
8. 2 µl of CFSE stain (cell viability stain) and 5 µl CD73/90 stain (MSC marker
stain) were added to 400 and 600 µl cell suspension in dPBS and incubated at
5 37ºC for 30min.
9. After 30 min incubation, cells were washed, centrifuged, and resuspended from
both the vials into 250 µl of PBS together.
10. 25 µl Opti-prep density gradient solution was added into the vial.
11. Cells were diluted by 1:10 ratio and flown into droplet generation device for 10 encapsulation.
12. Cells were immobilized into array device and imaged for CFSE, CD73 and
CD90 stains.
[0078] After staining the cells were washed, centrifuged (180 g, 5 min, 4ºC) and with gentle pipetting resuspended in media with 1/10th Opti-prep before droplet
15 encapsulation. 1 nanoliter sized droplets were generated which was sufficient to
allow cells to replicate at least 3-4 times (Figure 3A-B). These tiny droplets were spatially defined and lodged in nano-wells. In microfluidics platform (Droplet Microfluidic Platform), 1000 droplets are dispensed in a nano-well device. The cells were sorted out at 150 nanoliter/sec flow rate and imaging was done with automated
20 fluorescence microscope. The nano-wells on chip were physically separated to
prevent any cross talk and helpful to automated retrieval of selected cells. The microscope can automatically position the device to defined nano-well co¬ordinates. [0079] Bright-field and multiple fluorescence images were collected (Figure 3C).
25 The cell size (diameter), viability (CFSC) and surface markers (CD73/90) positivity
were analyzed. The analysis was done by artificial intelligence (AI) based tool trained for size-based identification of cells. After screening and imaging, the device was taken out and kept for incubation in a CO2 incubator until further processing. Image analysis was done with AI tool, which has been retrained to
30 identify cells in images. The model marked out the cell boundaries and locations
24

and provided the information of cell size and total fluorescence intensity. Programs
used in AI were developed in python using open-source standard libraries.
[0080] Size distribution and Mean fluorescence Intensity measurement – After
encapsulation by AI tool analysis, the varied size of the cells in droplets were
5 measured. Microscopy analysis confirmed distinct differences in cell size among
the encapsulated populations (Table 5). The average cell sizes were measured to be
12.2 ±1.9 µm, 19.6 ± 2.6 µm, 35.8 ± 6.7 µm for the small, medium-size, and large
MSC subpopulations, respectively. The small and large subpopulation each
contained 40 and 2% of the cells in total population, respectively. The majority was
10 sorted into medium size subpopulation, comprising 58% of the total cells.
Table 5: Segregation of three population based on size (diameter) and its equivalent mean fluorescence intensity (MFI) values of identification markers.

Population Size of cells (Avg ± SD) CD73 (MFI) (Avg ± SD) CD90 (MFI) (Avg ± SD)
Small 12.2 ±1.9 µm 5.67 ± 0.9 5.9 ± 0.8
Medium 19.6 ± 2.6 µm 6.0 ± 0.8 6.5 ± 0.7
Large 35.8 ± 6.7 µm 6.0 ± 0.15 6.8 ± 0.8
[0081] With the present exercise, the three subsets of populations (based on its size) were recognized where the expression of identification markers was measured. It
15 was hypothesized to obtain a correlation between mean fluorescence intensity
values with their respective cell size, however no significant changes were recorded
in this reference.
Retrieval of three subset populations
[0082] Once the cells were recorded, cells were retrieved with the AI tool for
20 automated size-based sorting and collected in vials containing 1ml PBS based on
their referenced size groups i.e., small, medium, and large. Immediately after
collection, cells were centrifuged at 180 g, 5 min, 4ºC and pellets were collected in
cultured DMEM media.
[0083] The similar microfluidics sorting and measuring was repeated 5-6 times in a
25 same day and cells were pooled. Approx 1000-3000 cells were there in each vial
and seeded in 96 well plate for further expansion. Media was changed in every 48
25

h. Once the optimum number of cells was achieved, the cells were passaged to
newer T25 flask followed to T75 and further expansion up was done using collagen
coated microcarriers (Cytodex 3) and bioreactor to scale up the cells up to P6 (cell
bank).
5 Example 3: Characterization of medium sized MSCs
Microarray Analysis [0084] After the confirmation about the qualified target medium sized population, the transcriptome of qualified cells was obtained via mRNA sequencing using Illumina platform. For microarray analysis, 500ng to 1000ng total RNA was
10 isolated from the 106 cells using Trizol lysis and measured using Qubit fluorometer,
in nuclease free water or low-TE (10mM Tris, 0.1mM EDTA). The parameter concentration ~50ng/µl with a minimum of 20µl of sample was fixed to get good yield. The RNA integrity number (RIN)≥8, with 2100 was screened with Bioanalyzer Un-degraded (DNase treated).
15 [0085] Library Preparation: A modified NEBNext RNA Ultra II directional
protocol was used to prepare the libraries for mRNA sequencing. In the first step the poly-A containing mRNA molecules were selectively purified using oligo-dT attached magnetic beads. Following purification, the mRNA was fragmented using divalent cations under elevated temperature. Next, the cDNA was synthesized using
20 Reverse transcriptase and random hexamers in a first strand synthesis reaction.
Subsequently, the cDNA was converted to double stranded cDNA where Uracil was added instead of Thymine. The strand specificity was preserved by a USER enzyme-based digestion of the second strand thereby leaving one functional strand, which maps to the DNA strand which it was transcribed from. The USER digested
25 single strand molecules were enriched and indexed in a limited cycle PCR followed
by AMPure bead purification to create final cDNA library for sequencing. Sequencing Prepared libraries were sequenced on Illumina HiSeqX/Novaseq to generate 2x150 bp reads/sample. Up to 75% of the sequenced bases were of Q30 value. Sequenced data were processed to generate FASTQ files and uploaded on
30 the FTP server.
Table 6: Information and data extracting with Microarray data.
26

Total Total Statistical Upregulated Upregulated (highly
Genes upregulated significance genes after expressed) genes in Upregulated
Identified genes criteria significant
criteria
(P≤0.001) Small sized population (highly expressed) genes in Medium sized population
57773 965 P≤0.001 110 34 64
Table 7: List of highly expressed genes in medium sized population with details.
27

S. NO Gene ID Gene symbol Ch r Start End Stra nd base Mean log2FoldC hange Small Sized Medium sized
1 ENSG00000 111799 COL12A1 chr 6 75794 042 75915 767 - 26780.9 3422 17.812254 64 0 53561.87
2 ENSG00000 115461 IGFBP5 chr 2 2.18E +08 2.18E +08 - 13548.3 8335 16.829192 76 0 27096.77
3 ENSG00000 186340 THBS2 chr 6 1.7E+ 08 1.7E+ 08 - 12835.6 8502 16.751234 19 0 25671.37
4 ENSG00000 166923 GREM1 chr 15 33010 175 33026 870 + 11399.2 204 16.580013 1 0 22798.44
5 ENSG00000 170558 CDH2 chr 18 25530 930 25757 410 - 6438.39 7316 15.755870 63 0 12876.79
6 ENSG00000 013297 CLDN11 chr 3 1.7E+ 08 1.71E +08 + 6056.94 7573 15.667761 96 0 12113.9
7 ENSG00000 133110 POSTN chr 13 38136 720 38172 981 - 6017.41 9103 15.658316 1 0 12034.84
8 ENSG00000 060718 COL11A1 chr 1 1.03E +08 1.04E +08 - 5557.30 7703 15.54356 0 11114.62
9 ENSG00000 140937 CDH11 chr 16 64977 656 65160 015 - 5120.12 2817 15.425354 98 0 10240.25
10 ENSG00000 108691 CCL2 chr 17 32582 304 32584 222 + 4925.24 7456 15.369374 05 0 9850.495
11 ENSG00000 003989 SLC7A2 chr 8 17354 597 17428 082 + 4537.47 3158 15.251069 61 0 9074.946
12 ENSG00000 144810 COL8A1 chr 3 99357 319 99518 070 + 4034.67 101 15.081635 37 0 8069.342
13 ENSG00000 152661 GJA1 chr 6 1.22E +08 1.22E +08 + 4011.74 4497 15.073414 24 0 8023.489
14 ENSG00000 091986 CCDC80 chr 3 1.12E +08 1.12E +08 - 3824.77 483 15.004560 88 0 7649.55
15 ENSG00000 163017 ACTG2 chr 2 74119 441 74146 992 + 3299.83 6738 14.791585 46 0 6599.673
16 ENSG00000 169604 ANTXR1 chr 2 69240 310 69476 459 + 3242.12 5171 14.766131 17 0 6484.25
17 ENSG00000 187720 THSD4 chr 15 71389 291 72075 722 + 3212.87 4103 14.753056 12 0 6425.748
18 ENSG00000 115963 RND3 chr 2 1.51E +08 1.51E +08 - 2208.45 5661 14.212241 4 0 4416.911
19 ENSG00000 101825 MXRA5 chr X 32266 06 32646 82 - 2200.94 5251 14.207326 9 0 4401.891
20 ENSG00000 182667 NTM chr 11 1.31E +08 1.32E +08 + 2177.22 8169 14.191696 59 0 4354.456
21 ENSG00000 187079 TEAD1 chr 11 12695 969 12966 298 + 2069.71 0729 14.118634 71 0 4139.421
22 ENSG00000 137693 YAP1 chr 11 1.02E +08 1.02E +08 + 2023.06 7133 14.085750 47 0 4046.134
23 ENSG00000 173546 CSPG4 chr 15 75966 663 76005 189 - 2007.65 1029 14.074715 03 0 4015.302
24 ENSG00000 255248 RP11-166D19.1 chr 11 1.22E +08 1.22E +08 - 1959.82 158 14.039929 58 0 3919.643
25 ENSG00000 146242 TPBG chr 6 83072 923 83080 545 + 1956.65 9302 14.037599 89 0 3913.319
26 ENSG00000 078098 FAP chr 2 1.63E +08 1.63E +08 - 1917.92 1401 14.008751 53 0 3835.843
28

27 ENSG00000 058085 LAMC2 chr 1 1.83E +08 1.83E +08 + 1742.41 4991 13.870299 18 0 3484.83
28 ENSG00000 139329 LUM chr 12 91496 406 91505 608 - 1580.74 3545 13.729817 23 0 3161.487
29 ENSG00000 074590 NUAK1 chr 12 1.06E +08 1.07E +08 - 1479.15 5376 13.633989 54 0 2958.311
30 ENSG00000 169213 RAB3B chr 1 52373 628 52456 436 - 1459.39 114 13.614582 94 0 2918.782
31 ENSG00000 164294 GPX8 chr 5 54455 946 54462 899 + 1453.06 6585 13.608317 29 0 2906.133
32 ENSG00000 134762 DSC3 chr 18 28569 974 28622 781 - 1428.95 4218 13.584176 68 0 2857.908
33 ENSG00000 128606 LRRC17 chr 7 1.03E +08 1.03E +08 + 1404.44 6566 13.559219 25 0 2808.893
34 ENSG00000 183098 GPC6 chr 13 93879 095 95059 655 + 1393.37 8594 13.547805 06 0 2786.757
35 ENSG00000 077942 FBLN1 chr 22 45898 118 45997 015 + 1362.54 6387 13.515523 78 0 2725.093
36 ENSG00000 154734 ADAMTS 1 chr 21 28208 066 28217 728 - 1336.45 7596 13.487633 08 0 2672.915
37 ENSG00000 185551 NR2F2 chr 15 96869 167 96883 492 + 1331.71 418 13.482503 6 0 2663.428
38 ENSG00000 112902 SEMA5A chr 5 90351 38 95461 87 - 1318.27 45 13.467870 24 0 2636.549
39 ENSG00000 145147 SLIT2 chr 4 20254 883 20622 184 + 1317.08 8645 13.466571 91 0 2634.177
40 ENSG00000 135111 TBX3 chr 12 1.15E +08 1.15E +08 - 1299.30 0834 13.446955 38 0 2598.602
41 ENSG00000 166173 LARP6 chr 15 71123 863 71146 498 - 1290.99 9855 13.437708 91 0 2582
42 ENSG00000 170961 HAS2 chr 8 1.23E +08 1.23E +08 - 1244.35 6259 13.384620 77 0 2488.713
43 ENSG00000 178882 FAM101 A chr 12 1.24E +08 1.25E +08 + 1240.00 8127 13.379570 86 0 2480.016
44 ENSG00000 185070 FLRT2 chr 14 85996 488 86095 034 + 1181.90 1275 13.310332 26 0 2363.803
45 ENSG00000 103855 CD276 chr 15 73976 307 74006 859 + 1168.46 1595 13.293833 39 0 2336.923
46 ENSG00000 176046 NUPR1 chr 16 28548 606 28550 495 - 1165.69 4602 13.290413 01 0 2331.389
47 ENSG00000 104067 TJP1 chr 15 29991 571 30261 068 - 1131.30 4833 13.247211 76 0 2262.61
48 ENSG00000 163629 PTPN13 chr 4 87515 468 87736 324 + 1111.54 0598 13.221785 21 0 2223.081
49 ENSG00000 072840 EVC chr 4 57129 24 58307 72 + 1087.42 823 13.190145 41 0 2174.856
50 ENSG00000 026559 KCNG1 chr 20 49620 193 49639 666 - 1038.80 8211 13.124155 91 0 2077.616
51 ENSG00000 149090 PAMR1 chr 11 35453 370 35551 848 - 974.376 804 13.031780 18 0 1948.754
52 ENSG00000 154553 PDLIM3 chr 4 1.86E +08 1.86E +08 - 947.892 7286 12.992025 01 0 1895.785
53 ENSG00000 159167 STC1 chr 8 23699 428 23712 320 - 942.358 7427 12.983577 76 0 1884.717
54 ENSG00000 168077 SCARA3 chr 8 27491 385 27534 293 + 930.104 9168 12.964695 23 0 1860.21
55 ENSG00000 116132 PRRX1 chr 1 1.71E +08 1.71E +08 + 872.393 3495 12.872282 43 0 1744.787
56 ENSG00000 063660 GPC1 chr 2 2.41E +08 2.41E +08 + 862.511 2318 12.855847 23 0 1725.022
57 ENSG00000 139874 SSTR1 chr 14 38677 204 38682 272 + 855.396 1071 12.843896 87 0 1710.792
29

58 ENSG00000 067048 DDX3Y chr Y 15016 019 15032 390 + 847.095 1282 12.829828 51 0 1694.19
59 ENSG00000 079931 MOXD1 chr 6 1.33E +08 1.33E +08 - 789.383 5609 12.728033 05 0 1578.767
60 ENSG00000 117152 RGS4 chr 1 1.63E +08 1.63E +08 + 773.176 8879 12.698105 69 0 1546.354
61 ENSG00000 121068 TBX2 chr 17 59477 257 59486 827 + 770.409 895 12.692933 52 0 1540.82
62 ENSG00000 128510 CPA4 chr 7 1.3E+ 08 1.3E+ 08 + 768.828 7561 12.689969 64 0 1537.658
63 ENSG00000 167779 IGFBP6 chr 12 53491 220 53496 129 + 767.642 902 12.687742 73 0 1535.286
64 ENSG00000 171724 VAT1L chr 16 77822 427 78014 004 + 754.203 222 12.662261 22 0 1508.406
Table 8: Importance of unique identified markers (Top 5) in medium sized population in reference to Autoimmune disease treatment:

Gene name Biologi cal Function Clinical correlation-with Arthritis & specific role Clinical correlation-with Type I Diabetes & specific role Clinical correlation-with multiple sclerosis & specific role Clinical correlation-with
inflammatory bowel disease & Sjogren’s syndrome & specific role
COL 12 A1 Skeletal system developm ent,
extracellu lar matrix structural constitue nt,
collagen fibril organizati on,
chondrog enic capacity • Clinical
(PMID: 27
701424)
• Specific
role
Enlisted in Chondro-specific genes; indicate MSCs inherent higher chondrogen ic capacity with efficient matrix organizatio n. -- • Clinical; in thyroid-associated ophthalmo pathy (PMID: 34 078122) --
IGF BP5 Matrix protein, Regulatio n of smooth muscle cell
migration ; and • IGF-1
Clinical correlate (PMID: 21 617253). With IGFBP5 -No report • No report
• Specific
role:
Could be
used as a
marker for
gestational • No report
• Specific
role:
IGFBP5 as an
inhibitory binding • Clinical;
(PMID: 24 379630)
30

regulation
of smooth
muscle
cell
proliferati
on • Specific role: As IGF-I modulator, it controls cartilage formation and
inhibiting degeneratio n i.e., impact on cartilage metabolism . diabetes mellitus protein for IGF-1 has role in amyotrophi c lateral sclerosis. Also plays crucial role in drive fibrosis and tissue remodeling with
Pulmonary fibrosis.
THB S2 Glycopr otein expressed in the extracellu lar matrix (ECM), role tissue remodelin g, potent inhibitor of tumor growth and
angiogen esis • Clinical
(PMID: 23 843355; PMID: 305 92390)
Specific role:
Marker to
predict
differentiation
potential of
MSCs for
cartilage
regeneration
therapy;
Endogenous
regulator of
angiogenesis
and
autoimmune
inflammation in
the synovium • Clinical
PMID: 363
35340;
PMID: 341
83428 Specific role: TSP2-deficient mice developed obesity and hyperglycemia. Mechanistically , high glucose increased activation of the hexosamine pathway and nuclear factor-κB signaling to elevate TSP2 expression. -- --
GRE M1 Membe r of the BMP (bone morphoge nic
protein) antagonis t family, role in regulating organoge nesis, tissue differenti ation inhibitor in the TGF-β • No report
Specific role:
Gremlin 1 outlines a resident of osteochondroret icular stem cells in the bone marrow -- -- --
31

signaling pathway
CDH • No report • Clinical; --- --
2 Also Specific role (PMID: 35
refer as NCAD, member of the cadherin superfami ly, plays role in formation of
cartilage and bone in cartilage regeneration & chondrogenesis predictability:
Mediates cell-cell interactions, regulates β-catenin (βcat) signaling, and promotes the chondrogenic differentiation of mesenchymal lineage cells 363774)
[0086] Sixty-four genes were found to be highly expressed in the medium-sized
MSCs as compared to small sized MSCs with 34 genes (Table 6 and Table 7; Figure
4). Among the 64 genes, the top five genes COL12 A1, IGFBP5, THBS2, GREM1
5 CDH2, were unique markers of medium sized population and also predicted to be
clinically associated with chondrogenesis and cartilage regeneration (Figure 5, Table 8) using the STRING online tool.
Cryopreservation and stability Studies of the medium-sized MSCs [0087] Around one billion expanded medium sized MSCs at the P6 stage were
10 cryopreserved in 3 sets of cryovials using medium containing 90% of FBS and 10%
DMSO (34 vials, 10 million MSCs each), 90% of HSA and 10% DMSO (33 vials, 10 million MSCs each), and 90% cord blood and maternal blood plasma along with10% DMSO (33 vials, 10 million, MSCs each) at a temperature below -150ºC in liquid nitrogen tank, under vapor phase conditions. After the completion of 6
15 month, 1 years, and 2 years, 1 vial from each sample was thawed at 37 ºC in water
bath and was further analyzed for Cell count, Cell viability, doubling time, growth kinetics, Annexin V, Cell Cycle analysis was performed to check the status of the cell stock. Table 9: Stability/Reprocess data (CB+MB plasma with DMSO)
32

Test Parameter Stability/Reprocess data at 6 months.
(Average value of 3 samples) Stability/Reprocess data at 1 year.
(Average value of 3 samples) Stability/Reprocess data at 2 years.
(Average value of 3 samples)
Cell Count 8.1 X10^6 ± 2.9 7.9 X10^6 ± 0.9 7.2 X10^6 ± 1.2
Cell Viability 95% 93% 91%
Annexin V 90.2 ± 2.3% 92.62 ± 1.3% 92.0 ± 1.8%
Cell Cycle G0/G1 phase G0/G1 phase G0/G1 phase
CD73/90/105 97.25 ± 1.1% 95.72 ± 2.7% 92.67 ± 3.0%
Cd3/19/56 0.32 ± 3.2% 0.43 ± 2.2% 0.60 ± 1.5%
CD34/45, HLADR 0.6% HLADR 0.7% HLADR 0.5% HLADR
Advantages of the present disclosure [0088] The present disclosure provides a method for producing MSCs with therapeutic potential with the following advantages.
a) The specific size (19.6 ± 2.6 µm), marker positivity (CD73/90/105) and gene
5 expression profile of the MSCs make them unique with significant therapeutic
potential.
b) The MSCs can be used as a cellular therapeutic tool for autoimmune disease
(like RA) treatment due to their potent suppressive action to inhibit
proinflammatory cells from both the innate and adaptive immune system.
10 c) The MSCs can be provided as off the shelf, allogenic therapy product.
d) The MSCs obtained using the disclosed method exhibits improved stability and viability even after cryopreserving up to 2 years.
e) The medium sized cells selected have shown higher expression of genes, which are responsible for the regeneration of cartilage, bone, etc for the tissue
15 regeneration process.
f) The microfluidics based MSCs cell sorting, and expansion thereafter gives
much more predictability for the end use of the cells in therapeutic applications.
33

We Claim:
1. A method of producing mesenchymal stem cells (MSCs) for therapeutic
application, the method comprising:
(a) culturing a heterogeneous population of MSCs in a culture medium for
5 a period in a range of 20 to 35 days;
(b) staining the MSCs to obtain a heterogeneous population of stained MSCs;
(c) adding droplet encapsulation media, comprising a density gradient solution to the heterogeneous population of stained MSCs, to obtain a
10 population of microfluidic droplets, wherein each droplet preferably
comprises a single MSC;
(d) providing the population of microfluidic droplets obtained from step (c)
to a microfluidics device; and
(e) identifying and selecting a population of homogeneous MSCs having
15 medium size in the range of 15 to 30 µm;
wherein the population of homogeneous MSCs obtained from step (e)
are viable MSCs, expressing MSC specific markers selected from CD
73, CD 90, and combinations thereof; and exhibiting increased
expression of genes selected from a group consisting of COL12A gene,
20 IGFBP5 gene, THBS2 gene, GREM1 gene, CDH2 gene, and
combinations thereof.
2. The method as claimed in claim 1, wherein the step (e) further comprises:
imaging the MSCs in the population of microfluidic droplets to record the
size of the MSCs; and subjecting the population of microfluidic droplets to
25 automated size-based sorting to obtain a population of homogeneous MSCs
having medium size.
3. The method as claimed in claim 1, wherein the step (a) further comprises:
reseeding the heterogeneous population of MSCs at a cell density in the
range of 5000 to 11,000/cm2 and expanding the heterogeneous population
30 of MSCs up to passage 3.
34

4. The method as claimed in claim 1, wherein the heterogeneous population of
MSCs in step (b) are stained using a cell viability stain or MSC marker stain;
and wherein the heterogeneous population of MSCs is having a
concentration in the range of 0.5 to 5x106 cells/ml, preferably having a
5 concentration of 1x106 cells/ml.
5. The method as claimed in claim 1, wherein the size of the microfluidic
droplet is in the range 100 to 200 micron; and each of the microfluidic droplet
is sufficient to allow the MSCs to replicate 3 to 4 times.
6. The method as claimed in claim 1, wherein the population of microfluidic
10 droplets are produced using a droplet generation device.
7. The method as claimed in claim 1, wherein the step (d) further comprising,
analyses of the microfluidic droplets at a flow rate in the range of 100 to 200
nanolitre/sec, preferably at a flow rate of 150 nanolitre/sec.
8. The method as claimed in claim 1, wherein the method further comprises,
15 expanding the population of homogeneous MSCs having medium size up to
passage 6 or up to passage 20 in culture; and optionally cryopreserving the MSCs.
9. The method as claimed in claim 4, wherein the cell viability stain is selected
from carboxyfluorescein diacetate succinimidyl ester (CFSE), trypan blue,
20 propidium iodide (PI), or combinations thereof; and the MSC marker stain
is selected from CD73, CD90, CD105, or combinations thereof.
10. The method as claimed in claim 1, wherein the culture medium is selected
from Dulbecco’s Modified Eagle Medium (DMEM), Iscove's Modified
Dulbecco's Medium (IMDM), DMEM-F12, F12, Minimum Essential
25 Medium α (ALPHA-MEM), or combinations thereof.
11. The method as claimed in claim 1, wherein the heterogeneous population of
MSCs is derived from human umbilical cord tissue of single donor or
multiple donors.
12. The method as claimed in claim 11, wherein the heterogeneous population
30 of MSCs is obtained by treating the human umbilical cord tissue with at least
one enzyme selected from collagenase, hyaluronidase, proteins identified
35

from CB+MB plasma lysate (Peroxiredoxin 1- PRDX1 and Heat Shock Protein-70 -HSP70), or mixtures thereof.
13. The method as claimed in claim 2, wherein the automated size-based sorting
is performed using an artificial intelligence-based tool trained for size-based
5 identification of cells.
14. The method as claimed in claim 1, wherein the homogeneous population of MSCs comprises 58% to 60% of MSCs having medium size.
15. A composition comprising the MSCs obtained by the method as claimed in claim 1; and an excipient; wherein the excipient is selected from DMEM,
10 human serum albumin (HSA), dimethyl sulfoxide (DMSO), fetal bovine
serum (FBS), cord blood and maternal blood plasma or combinations thereof.
16. A formulation comprising the MSCs obtained by the method as claimed in
claim 1, or the composition as claimed in claim 15; and a pharmaceutically
15 acceptable carrier.
17. The formulation as claimed in claim 16, wherein the pharmaceutically
acceptable carrier is selected from ringer lactate solution, saline, dextrose,
heparin, or combinations thereof.
18. A method of treating a disease in a subject, comprising administering the
20 MSCs obtained from the method as claimed in claim 1 or the formulation as
claimed in claim 16 to a subject.
19. The method as claimed in claim 18, wherein the MSCs is allogenic to the
subject.
20. The method as claimed in claim 18, wherein the disease is selected from a
25 group consisting of autoimmune diseases (Rheumatoid Arthritis (RA),
Acquired Aplastic Anemia, Acquired Hemophilia, Agammaglobulinemia,
Alopecia Areata, Ankylosing Spondylitis (AS), Anti-NMDA Receptor
Encephalitis, Antiphospholipid Syndrome (APS), Arteriosclerosis,
Autoimmune Addison’s Disease (AAD), Autoimmune Autonomic
30 Ganglionopathy (AAG), Autoimmune Encephalitis (AE)/Acute
Disseminated Encephalomyelitis (ADEM), Autoimmune Gastritis,
36

Autoimmune Hemolytic Anemia (AIHA), Autoimmune Hepatitis,
Autoimmune Hyperlipidemia, Autoimmune Hypophysitis/Lymphocytic
Hypophysitis, Autoimmune Inner Ear Disease (AIED), Autoimmune
Lymphoproliferative Syndrome (ALPS), Autoimmune Myelofibrosis
5 (AIMF), Autoimmune Myocarditis, Autoimmune Oophoritis, Autoimmune
Pancreatitis (AIP), Autoimmune Polyglandular Syndromes (APS),
Autoimmune Progesterone Dermatitis (APD), Autoimmune Retinopathy
(AIR), Autoimmune Sudden Sensorineural Hearing Loss, Balo
Disease/Concentric Sclerosis, Behçet’s Disease, Birdshot
10 Chorioretinopathy/Birdshot Uveitis, Bullous Pemphigoid, Castleman
Disease, Celiac Disease, Chagas Disease, Chronic Inflammatory
Demyelinating Polyneuropathy (CIDP), Chronic Autoimmune Urticaria,
Churg-Strauss Syndrome/Eosinophilic Granulomatosis with Polyangiitis
(EGPA), Cogan's Syndrome (CS), Cold Agglutinin Disease (CAD), Crest
15 Syndrome, Crohn's Disease, Stricturing Crohn's Disease, Cronkhite-Canada
Syndrome (CCS), Cryptogenic Organizing Pneumonia (COP), Dermatitis
Herpetiformis (DH), Dermatomyositis, Diabetes, Type 1 (T1D), Discoid
Lupus Erythematosus (DLE), Dressler’s Syndrome/ Post myocardial
Infarction/ Post pericardiotomy Syndrome, Eczema/Atopic Dermatitis,
20 Eosinophilic Fasciitis, Erythema Nodosum, Essential Mixed
Cryoglobulinemia, Evans Syndrome, Fibrosing Alveolitis/Idiopathic
Pulmonary Fibrosis (IPF), Giant Cell Arteritis/Temporal Arteritis/Horton's
Disease, Giant Cell Myocarditis, Glomerulonephritis (GN), Goodpasture’s
Syndrome/Anti-Gbm/Anti-Tbm Disease, Granulomatosis With Polyangiitis
25 (GPA)/Wegener's Granulomatosis, Graves’ Disease (GD), Guillain-Barrè
Syndrome (GBS), Hashimoto’s Thyroiditis/Autoimmune Thyroiditis,
Henoch-Schölein Purpura (HSP)/Iga Vasculitis, Hidradenitis Suppurativa,
Hurst’s Disease/Acute Hemorrhagic Leukoencephalitis (AHLE),
Hypogammaglobulinemia, Iga Nephropathy/Berger’s Disease, Immune-
30 Mediated Necrotizing Myopathy (IMNM), Immune Thrombocytopenia
(Itp)/Autoimmune Thrombocytopenia Purpura, Inclusion Body Myositis
37

(IBM), Igg4-Related Sclerosing Disease (ISD), Interstitial Cystitis, Juvenile
Idiopathic Arthritis (Jia)/Adult-Onset Still's Disease, Juvenile
polymyositis/Juvenile dermatomyositis/ juvenile myositis, Kawasaki
disease, Lambert-Eaton Myasthenic Syndrome (LEMS), Leukocytoclastic
5 vasculitis, Lichen Planus, Lichen Sclerosus, Ligneous conjunctivitis, Linear
Iga Disease (LAD), Lupus Nephritis (LN), Lyme Disease /Chronic Lyme
Disease/Post-Treatment Lyme Disease Syndrome (PTLDS), Lymphocytic
colitis/microscopic colitis, Lymphocytic hypophystitis /autoimmune
hypophystitis, Ménière’s Disease, Microscopic Polyangiitis (MPA)/ANCA-
10 Associated Vasculitis, Mixed Connective Tissue Disease (MCTD),
Mooren’s ulcer, Mucha-Habermann disease, Multifocal motor neuropathy,
Multiple Sclerosis (MS), Myalgic Encephalomyelitis/Chronic Fatigue
Syndrome (ME/CFS), Myasthenia Gravis (MG), Narcolepsy, Neuromyelitis
Optica/Devic's Disease, Ocular Cicatricial Pemphigoid, Opsoclonus-
15 myoclonus syndrome (OMS), Palindromic Rheumatism, Paraneoplastic
Cerebellar Degeneration (PCD), Paraneoplastic Pemphigus, Parry-Romberg
Syndromeherth (PRS)/Hemifacial Atrophy (HFA)/Progressive Facial
Hemiatrophy, Paroxysmal Nocturnal Hemoglobinuria (PNH), Peripheral
uveitis/pars planitis, PANS/PANDAS, Parsonage-Turner Syndrome (PTS),
20 Pemphigoid Gestationis (PG), Pemphigus Foliaceus, Pemphigus Vulgaris,
Pernicious anemia, POEMS Syndrome, Polyarteritis Nodosa (PAN),
Polymyalgia Rheumatica, Polymyositis, Postural Orthostatic Tachycardia
Syndrome (Pots), Primary Biliary Cirrhosis (PBC), Primary Sclerosing
Cholangitis (PSC), Psoriasis, Palmoplantar Pustulosis (PPP), Psoriatic
25 Arthritis, Pulmonary fibrosis, idiopathic (IPF), Pure Red Cell Aplasia
(PRCA), Pyoderma gangrenosum, Rasmussen's encephalitis, Raynaud’s
Syndrome, Reactive Arthritis, Reflex sympathetic dystrophy syndrome
(RSD)/Complex regional pain syndrome (CRPS), Relapsing Polychondritis
(RP), Restless leg syndrome (RLS)/Willis-Ekbom disease, Rheumatic Fever,
30 Sarcoidosis, Schmidt Syndrome/Autoimmune Polyendocrine Syndrome
Type II, Scleritis, Scleroderma, Sclerosing Mesenteritis/Mesenteric
38

Panniculitis, Serpiginous choroidopathy, Sjögren's Syndrome, Stiff person
syndrome (SPS), Small Fiber Sensory Neuropathy (SFSN), Small Fiber
Sensory Neuropathy (SFSN), Systemic Lupus Erythematosus (SLE),
Subacute bacterial endocarditis (SBE), Subacute cutaneous lupus, Susac’s
5 syndrome, Sydenham’s Chorea, Sympathetic ophthalmia, Takayasu’s
arteritis (vasculitis), Testicular Autoimmunity, Tolosa-Hunt syndrome,
Transverse myelitis (TM), Tubulointerstitial nephritis uveitis syndrome
(TINU), Ulcerative Colitis, Undifferentiated Connective Tissue Disease,
Uveitis, Vasculitis, VEXAS Syndrome, Vogt-Koyanagi-Harada syndrome
10 (VKH), Osteoarthritis, AVN, vertebral compression factor, urethral stricture,
Sjogren’s syndrome and ureteric stricture), arthritis, Type I Diabetes, multiple sclerosis, inflammatory bowel diseases and acromegaly.