CN120053618A

CN120053618A - Application of human iPSC platelet-rich suspension in arthritis treatment

Info

Publication number: CN120053618A
Application number: CN202311628440.7A
Authority: CN
Inventors: 王文元
Original assignee: Heluo Xintu Biotechnology Shanghai Co ltd
Current assignee: Heluo Xintu Biotechnology Shanghai Co ltd
Priority date: 2023-11-30
Filing date: 2023-11-30
Publication date: 2025-05-30

Abstract

The invention provides a composition for treating arthritis, comprising a platelet concentrate prepared by differentiation of human pluripotent induced stem cells (Inducedpluripotent stem cell, iPSC) and an additive for preparing a platelet suspension or gel. The invention aims to provide a functional platelet-rich composition which can be prepared in large scale, is stable in batches and can be produced in a standardized way in the aspect of treating arthritis. Platelet concentrate and blood-separated Platelet-rich plasma (PRP) in the composition of the invention are completely different in composition, are relatively stable in composition, and are helpful for tissue repair by slow release of growth factors.

Description

Application of human iPSC platelet-rich suspension in arthritis treatment

Technical Field

The invention relates to the field of biomedical materials, in particular to an application of an iPSC in-vitro preparation of a platelet-rich material in osteoarthritis.

Background

Arthritis (arthritis) is of over 100 different types and has different etiologies and treatment regimens. Of these, osteoarthritis (OA) and rheumatoid arthritis (rh um a t omicron ID ARTHRITIS, RA) are the two most common arthritic conditions. Osteoarthritis is a degenerative disease that causes articular cartilage fibrosis, chapping, ulceration, and loss due to various factors, and is mainly manifested by joint pain. About 2.4 million people worldwide are affected by this, 50% of people over 65 years old are osteoarthritis patients. Rheumatoid arthritis is a complex disease associated with multiple factors of systemic autoimmunity, which is mainly characterized by erosive arthritis. The global prevalence of rheumatoid arthritis is about 1% and the ratio between men and women is about 1:3.

The main pathological characteristics of osteoarthritis are degradation and degeneration of articular cartilage, remodeling of subchondral bone, synovial membrane and bone spur hyperplasia, etc. OA pathogenesis is complex in that cartilage degeneration involves cross-regulation between multiple pathways and phenotypes, such as MAPK pathway, NF- κb pathway, apoptosis, autophagy, anabolism and catabolism, etc., and thus is extremely difficult to cure. The main pathological features of rheumatoid arthritis are joint synovitis, immune cell infiltration and pannus formation leading to sustained destruction of cartilage and subchondral bone.

Biological agents rich in high concentrations of platelets and growth factors, such as platelet-rich plasma (PRP), have excellent tissue repair and regeneration potential. PRP can stimulate cartilage regeneration, increase hyaluronic acid production, promote formation of new blood vessels in the joint, and thereby increase the amount of nutrients reaching the damaged area of articular cartilage. Studies have shown that platelet-derived growth factors IGF-1 (insulin-like growth factor 1) and TGF-beta (transforming growth factor beta) play a key role in chondrocyte differentiation, collagen synthesis and matrix deposition. IGF-1 inhibits NF- κB signaling through regulation of MAPK and PI3K/Akt signaling pathways and inhibits apoptosis by reducing ROS production, as in the rabbit knee osteoarthritis model, thereby protecting chondrocytes. In addition, mouse studies indicate that TGF- β/TAK1-FoxO1 cascade signaling pathway can regulate articular cartilage autophagy and homeostasis maintenance.

Although Platelet-rich plasma (PRP) is currently used in a variety of clinical treatment directions, such as orthopedics (bone injury repair, cartilage injury repair, etc.), sports injury (acute sports injury, chronic mechanical injury, etc.), general external directions (acute trauma, chronic difficult-to-heal wound treatment, etc.), burn plastic directions (burn wound, skin tissue regeneration, skin adipose tissue liquefaction, etc.), and dental directions (periodontal disease, jaw surgery, etc.), among others. However, since it is generally prepared by autologous blood drawing, it is often limited and influenced by donor background and sampling amount, and due to the diversification of individual blood quality and preparation method (currently, there is no unified standard preparation flow), the previous data cannot generate widely and effectively guidance for clinic, which is clinically dependent on hospital system and doctor experience. In addition, since PRP is prepared in vitro, some risks associated with infection and other operations are also involved.

The preparation of iPSC-platelets is a prerequisite for the production of a homogeneous platelet concentrate product, as well as for the production of corresponding therapeutic standards. The preparation of the iPSC platelets is realized through in vitro differentiation of the iPSC, and the in vitro differentiation and regulation path of the whole process is clear at present. In 2018, published by Koji Eto group of Kyoto university in Japan, by using an 8L turbulent platelet reactor, 1X 10 ¹¹ platelets can be produced at one time, the production efficiency of 70-80 platelets produced by 1 megakaryocyte is realized, and a foundation is laid for producing platelets in vitro.

However, in the aspect of treating arthritis, the prior art still lacks functional platelets which can be prepared in large quantity, stabilize the quality of each batch and can be produced in a standardized way.

Disclosure of Invention

The present invention provides a method of in vitro differentiation and platelet production and in vitro activation by human pluripotent induced stem cells (ipscs, induced pluripotent stem cell) using a reactor, and finally provides a platelet composition for the treatment of osteoarthritis or rheumatoid arthritis.

In order to achieve the above purpose, the present invention provides the following technical solutions:

the invention provides a composition for treating arthritis, which comprises the following components:

(1) Platelet concentrates prepared by multipotent induced stem cell differentiation;

(2) The additive comprises physiological saline with 0.9% NaCl and 5% recombinant human serum albumin, wherein the administration dosage of the composition is 5mL.

Preferably, the above-mentioned supplement further comprises 100U/mL thrombin lyophilized powder, 1% calcium gluconate and 0.05% type I collagenase.

The invention provides a composition for treating arthritis, which is characterized by comprising the following components:

(2) The additive comprises 10 mu M ADP, 10-100 mu g/mL fibrinogen, 2mM CaCl ₂ and 0.05-0.5% type I collagenase, wherein the administration dosage of the composition is 5mL.

Preferably, the concentration of the platelet concentrate is 5-20X 10 ¹¹/L.

Preferably, the frequency of administration of the above composition and the above gel is 1 time per week, with 3 times each being a course of treatment.

More preferably, the use of the above composition in the manufacture of a medicament for the treatment of arthritis.

Compared with the prior art, the invention has the beneficial effects that:

(1) The differentiation process of iPSC into hematopoietic precursor stem cells is divided into three stages, and each stage has its own corresponding culture medium formulation, which helps to improve the purity of CD34 ⁺ cells.

(2) In the process of differentiating hematopoietic precursor stem cells into megakaryocytes, 50ng/mL IL-21, 5nM Tazemetostat, 200nM Eltrombopag and 200nM iBET151 are added to the medium to promote differentiation and proliferation of megakaryocytes.

(3) After cryopreservation and resuscitation of megakaryocytes, and prior to use of the reactor, 0.5ng/mL collagen, 5nM Fingolimod HCl, was added to help promote platelet maturation.

(4) The platelet-producing reactor has a high density grid structure that acts to cut the fluid stream, can produce severe megakaryocyte fragmentation and helps to release platelets.

(5) Compared with the PRP separated by blood, the iPSC platelet preparation is stable in composition, and the slow-release growth factors are beneficial to tissue repair.

(6) The preparation methods (suspension formulation, activated suspension preparation formulation, gel preparation formulation and preparation method) are never used in the application scene of iPSC-blood platelets.

(7) Can efficiently produce platelet-derived growth factors, has stable property and batch, can produce effective treatment effects and can produce referenceable clinical data for arthritis treatment.

Drawings

The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this specification, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

FIG. 1 shows the morphology (a) of hematopoietic precursor stem cells (Hematopoietic progenitor cells) produced by pluripotent induced stem cells (iPSC), the cell size (b) of hematopoietic stem cells, and the CD34 ⁺ cells (c) obtained by passaging purification and their purity (d). CD34, a marker expressed primarily in hematopoietic stem cells and hematopoietic progenitor cells. BF bright field, bright field.

Fig. 2 is a comparison of data after cryopreservation and resuscitation of megakaryocytes (iMKs) derived from iPSC source (a) from neonatal male (a) and 34 year old female (B), respectively, including morphology (B, c), proliferation capacity (d, e), cell viability (f), and cell size (g).

FIG. 3 shows the in vitro maturation and shedding process of platelets (a), and the proportion of platelets produced by single megakaryocytes (b), the overall size of platelets (c) in static and reactor environments, and the morphology observation shows that intense megakaryocyte fragmentation and platelet release (d) can occur in the reactor for 72 hours. By flow cytometry, iPSC-derived platelets (iPLTs) were found to have similar marker expression (f) compared to peripheral blood-derived platelets (e). BF bright field, bright field. PB-PLTS, peripheral blood derived platelets.

FIG. 4 shows the state of the platelet suspension during the preparation of the platelet suspension after the deactivation and activation.

Detailed Description

The present invention will be further described with reference to the accompanying drawings and specific examples, to which the invention is not limited. It is also to be understood that the terminology used in the examples of the invention is for the purpose of describing particular embodiments only, and is not intended to be limiting of the scope of the invention. Variations and advantages that will occur to those skilled in the art are included within the following claims and any equivalents thereof without departing from the spirit and scope of the inventive concept.

All technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. In other instances, certain terms used herein will have their meaning set forth in the specification. The experimental methods in the following examples, in which specific conditions are not specified, are common knowledge and common knowledge to those skilled in the art. The embodiments of the present application and the features of the embodiments may be combined with each other.

A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification taken in conjunction with the drawings. The examples provided are merely illustrative of the methods of the present invention and are not intended to limit the remainder of the disclosure in any way whatsoever.

In order to provide a platelet preparation with stable components and a preparation method which can be prepared in a large amount, is stable in batches and can be produced in a standardized way, the invention achieves technical effects by using an iPSC platelet preparation method, a reactor, a suspension formula, an activated suspension formula, a gel formula and a preparation method.

The invention provides a method for preparing platelets by using stem cells. First, the process of differentiating pluripotent induced stem cells (ipscs) into hematopoietic precursor stem cells (HPCs) is divided into three phases, with each phase having its own corresponding medium formulation:

Table 1.

Next, in the process of differentiating HPC Into Megakaryocytes (iMK), 50ng/mL IL-21, 5nM Tazemetostat, 200nM Eltrombopag, 200nM iBET151 was added to the medium to promote differentiation and proliferation of megakaryocytes.

Finally, after cryopreservation and resuscitation of megakaryocytes, a maturation process from static to dynamic (see example 3, points 1 and 2) was added, and platelet maturation was promoted by adding 0.5ng/mL Collagen (Collagen) and 5nM Fingolimod HCl.

The present invention uses a reactor to produce platelets, wherein the reactor has a high density grid structure that acts to cut fluid flow in two ways, 1) grid static and fluid dynamic, or 2) grid dynamic and fluid static.

Although the iPSC platelet preparation method of the present invention refers to the conventional PRP preparation concept, the iPSC platelet preparation and the blood-separated PRP are completely different in composition, and the blood-separated PRP composition is relatively unstable. The traditional iPSC blood platelet comprises a large proportion of plasma components, and the components and the proportion of the traditional iPSC blood platelet have large differences due to different sources of individuals and blood banks, so that the effect difference in the application process can be caused, the components of the iPSC-blood platelet are determined, the batch is stable, the component difference caused by different sampled individuals is avoided, the traditional iPSC blood platelet can be prepared on a large scale and is strictly controlled in quality, and the standardized product with strictly controlled production process and product quality is obtained. Thus, the dosage and the effect of clinical application can be well guided and referred. Accordingly, the iPSC-derived platelet rich suspension product is different from peripheral blood PRP, which does not contain white blood cell components and plasma components. The platelet-rich suspension from the iPSC contains the components of iPSC induced platelets and 5% albumin, and the platelet growth factors which are generated in a large amount after activation can play a role in repairing tissue injury, especially form gel PRP, and the components such as the platelets, the growth factors and the like are entangled by fiber interweaving, so that a slow release effect can be achieved, a relatively long-term use effect is achieved, and the effect on tissue repair is also achieved.

The suspension and gel additive formulations of the present invention are as follows:

Table 2.

In addition, the iPSC platelet preparation can be applied to different scenes, such as arthritis and tennis elbow treatment, wound healing promotion, alopecia treatment and the like, and can also be applied to the field of cosmetology.

Example 1 differentiation of iPSC into hematopoietic precursor Stem cells (Hematopoietic progenitor cells, iHPCs)

The iPSC of the invention is a hospital skin biopsy sample, primary fibroblasts are cultured in a laboratory, and an ideal iPSC cell line is obtained by screening after reprogramming.

1. IPSC was dissociated into single cells, plated on a culture dish pre-packed with human recombinant laminin (RHLAMININ-521, thermo-filter) and cultured for 2 days in the medium of Essential 8 (Thermofisher) with 30ng/mL VEGF-A165, 10ng/mL bFGF, 5. Mu. M Y-27632, 2mM Chir-99021 and 20ng/ML ACTIVIN A.

2. The medium was then replaced with HDM as follows:

HDM Medium Iscove Modified Dulbecco (IMDM) 1×ITS, 1×glutamax, 0.45: 0.45mM monothioglycerol, 50g/mL ascorbic acid and 20% KO-SRM were added.

Thereafter, 30ng/mL VEGF-A165, 10ng/mL bFGF, 20ng/mL BMP4, 5. Mu. M Y-27632 and 20ng/ML ACTIVINA were added and the culture was continued for 4 days.

3. Thereafter, the medium was changed to HDM medium, and 30ng/mL VEGF-A165, 10ng/mL bFGF, 50ng/mL Stem Cell Factor (SCF), 50ng/mL Thrombopoietin (TPO), 2U/mL Erythropoietin (EPO) and 5 mu M Y-27632 were added, and further 50ng/mL IL-3, 50ng/mL IL-6 were optionally added, and the culture was continued for 8 days.

4. Hematopoietic precursor stem cells (iHPCs) grown in suspension were collected, and the culture supernatant containing iHPCs was collected and centrifuged at 1000 rpm/min for 5 minutes.

The results are shown in FIG. 1. The hematopoietic precursor stem cells (Hematopoietic progenitor cells, HPC) produced by ipscs of this example (fig. 1 a) had a cell size similar to CD34 ⁺ cells isolated from peripheral blood (fig. 1 b), and the purity of the resulting CD34 ⁺ cells (fig. 1 c) could reach about 90% by passaging purification (fig. 1 d), with more starting material helping to reduce the interference of the subsequent differentiation process and without generating too many heterogeneous cells.

EXAMPLE 2 differentiation and proliferation of megakaryocytes

I. differentiation of hematopoietic precursor stem cells (iHPC) Into Megakaryocytes (iMKs)

1. The hematopoietic precursor stem cells obtained in example 1 were collected, plated on a culture dish pre-packed with 0.1% gelatin, and 20ng/mL VEGF-A165, 5ng/mL bFGF, 50ng/mL Thrombopoietin (TPO), 20ng/mL IL-3, 20ng/mL IL-6, 50ng/mL IL-21, 5nM Tazemetostat, 200nM Eltrombopag and 200nM iBET151 were added to the HDM medium and the culture was continued for 15 to 20 days.

2. The suspended megakaryocytes were collected.

In vitro proliferation of megakaryocytes (iMKs)

1. Transferring megakaryocyte obtained in the step I, placing the megakaryocyte in a culture flask/shake flask made of low-adsorption materials, replacing culture solution with HDM, adding 50ng/mL Thrombopoietin (TPO), 50ng/mL Stem Cell Factor (SCF), 50ng/mL IL-21 and 5 mu M Y-27632, placing the mixture on a horizontal shaking table, and amplifying, and regulating the rotating speed to 120-150 rpm.

2. Megakaryocytes can be expanded in vitro for several months while maintaining their properties, with a passaging period of 3 days and a seed density of 5X 10 ⁶/mL each time.

3. Megakaryocytes can be cryopreserved, the stock solution is HDM+5% BSA+5% DMSO, and the megakaryocytes are stored in liquid nitrogen after programmed freezing.

The results are shown in FIG. 2. Megakaryocytes (iMKs) derived from two different background ipscs (fig. 2 a) of neonatal male (a) and 34 year old female (B), respectively, can maintain a dispersed uniform morphology (fig. 2B, fig. 2 c), a proliferation capacity of 15-ten thousand times (fig. 2d, fig. 2 e), and a relatively stable cell survival rate (fig. 2 f) and cell size (fig. 2 g) after cryopreservation and resuscitation.

Example 3: platelet production

1. Megakaryocytes obtained in example 2 were suspended in PM medium, 50ng/mL Thrombopoietin (TPO) and 5. Mu. M Y-27632 were added, and the mixture was allowed to stand for 1 day.

PM medium Iscove Modified Dulbecco (IMDM) 1x ITS,1x glutamax, 0.45mM thioglycerol (monothioglycerol), 50g/mL ascorbic acid (ascorbic acid), 10U heparin (heparin), 5% human plasma (human plasma) were added.

2. Changing the culture medium to PM culture medium, adding 50ng/mL Thrombopoietin (TPO), 0.5ng/mL Collagen (Collagen), 5nM Fingolimod HCl and 5 mu M Y-27632, standing for 1 day, continuously culturing on a horizontal shaking table for 1 day, and regulating the rotating speed to 120-150 rpm.

3. Transferring megakaryocytes obtained in the steps into a bioreactor, performing platelet maturation in the reactor of a 1L system, and continuously culturing for 5-6 days, wherein the culture environment is 37 ℃ and 5% CO ₂, and the maximum horizontal liquid flow linear speed in the reactor is 30 cm/s.

The reactor has the specifications of cylindrical appearance, external tank height of 160mm, bottom diameter of 130mm and culture container thickness of 5mm, embedded multilayer movable grid structure, variable direction rotary motion mode, rotation speed of 120-150 rpm and maximum linear liquid flow speed of 30 cm/s.

The results are shown in FIG. 3. In the in vitro maturation and shedding process of platelets (fig. 3 a), in parallel control of static and reactor environments, the proportion of platelets produced by single megakaryocytes in the reactor conditions can reach more than 1:100 (fig. 3 b), and the overall size is closer to that of platelets derived from peripheral blood (fig. 3 c), and morphological observation shows that severe megakaryocyte fragmentation and platelet release can be produced in the reactor for 72 hours (fig. 3 d). By flow cytometry, iPSC-derived platelets (iPLTs) were found to have similar marker expression (fig. 3 f) compared to peripheral blood-derived platelets (fig. 3 e).

Example 4 preparation of platelet suspensions and gels

1. Human iPSC-derived platelet-rich suspension

The iPSC platelets of example 3 were concentrated and purified, and then suspended in physiological saline containing 0.9% NaCl at a concentration of 5 to 20X 10 ¹¹/L, to which 5% human recombinant serum albumin (rhHSA) was added.

2. Activated human iPSC-derived platelet-rich suspension

100U/mL thrombin lyophilized powder, 1% calcium gluconate and 0.05% type I collagenase were added on the basis of the suspension at point 1 above.

3. Human iPSC-derived platelet-rich gel

The iPSC platelets obtained in example 3 were concentrated to 1X 10 ¹¹/L or more, suspended in PBS, 10. Mu.M ADP, 10 to 100. Mu.g/mL fibrinogen (Fibrinogen), 2mM CaCl ₂ and 0.05 to 0.5% type I collagenase were added to the solution, and the solution was placed in a room temperature sterile environment and allowed to stand for crosslinking for 5 minutes. Centrifuging at 200rpm for 15 min, centrifuging at 1500rpm for 2 min, and discarding supernatant to obtain the lower layer gel rich in activated platelets.

The results are shown in FIG. 4. During the preparation of the platelet suspension, the unactivated platelet suspension (described in example 1. Method) exhibited a clearer state, and after activation (described in example 2. Method), the suspension became turbid immediately due to platelet aggregation and fibrin entanglement (the control group was peripheral blood isolated platelets). Wherein, compared with the peripheral blood of the control group, the platelet is separated, and the aggregation effect of the iPSC platelets is better.

Example 5 platelet suspension for use in the treatment of arthritis

1. The preparation method of the platelet rich suspension derived from human iPSC is as in examples 1-4.

2. The platelet suspension is used for treating arthritis, particularly osteoarthritis and rheumatoid arthritis.

3. The injection mode is that the knee joint is injected in the cavity under the guidance of ultrasound, a 22-gauge needle is used, 5mL of fresh human iPSC source platelet-rich suspension is used each time, the knee joint is passively flexed and stretched for 5 times after the injection, and the patient takes a rest for 10 minutes. Patients were followed up within 12 months after injection 1 time per week, 3 times per course of treatment.

4. The evaluation method for treatment improvement is 1) average knee pain score (11-point digital score, 0=no pain-10 most severe pain), 2) MRI measurement of medial cartilage volume of tibia, 3) 31 auxiliary evaluation indexes (25 symptom correlations, 6 MRI evaluations) to evaluate pain, function, quality of life, overall change, joint structure, etc.

Example 6 application of platelet suspension for the treatment of tennis elbow

2. The platelet suspension described above is used for the treatment of tennis elbow.

3. The treatment mode comprises the steps of sealing an injection part by 0.5% bupivacaine and epinephrine, injecting 2-3 mL of the prepared human iPSC source platelet-rich suspension into the extensor carpi radialis tendon and surrounding area by a tendon puncture method, and performing penetration by 1-time puncture without pulling out and switching 5-time direction injection tendon.

4. The treatment improvement was evaluated by 1) 4, 8, 12, 16, 20, 24 weeks after treatment, an anti-wrist extension VAS pain score (assessment of pain severity by visual simulation, VASRWE) 25% or more improvement in pain score over baseline was considered successful, 2) an auxiliary evaluation index, patient scoring tennis elbow assessment questionnaire (PRTEE), and an extended wrist examination.

EXAMPLE 7 platelet suspension application to wound healing treatment

2. The suspension can be used for promoting wound healing, such as wound and burn healing.

3. The treatment mode is that after the trauma is treated by surgery, the platelet rich suspension from the iPSC source is applied in the wound before closing the suture incision, the concentration of platelets is not lower than 1X 10 ⁶/mu L at each site, and the dosage is adjusted according to the area of the trauma and the damage degree.

4. The evaluation indexes of treatment improvement are 1) wound healing time, 2) wound infection evaluation and 3) wound healing quality.

Typically wounds use inactive platelet PRP to allow slow release of growth factors to promote healing, whereas wounds that are severely damaged or prone to infection use active platelets to allow a large amount of growth factors to be enriched and rapidly released from the wound in a short period of time to rapidly promote wound healing.

Example 8 platelet suspension for cosmetic application

2. The suspension is used in the cosmetic field.

3. The treatment mode comprises direct injection (dermis layer, subcutaneous shallow layer), smearing and combined use, and can be used for 1-2 months for 1 time and 3-4 times for each treatment course according to different application directions.

4. Treatment notes:

The treatment of human iPSC-derived platelet-rich suspensions does not require a recovery period and can be normally performed the next day after treatment. Swelling or bleeding points in the treated area may occur after treatment, and typically slight swelling may be eliminated on the day, and bleeding points may be eliminated typically 2-3 days. Note that:

(1) Women will prepare and use concentrated platelet preparations during non-menstrual periods. Patients with pregnancy, lactation, female with pregnancy, anemia, coagulation dysfunction, and liver dysfunction are forbidden;

(2) The treatment area is kept dry 24 hours after treatment, no water is adhered, no other skin care products with stimulation are used, sun protection is noted, anticoagulant drugs are not taken 2 weeks before treatment, and sweat steaming and massage are not recommended 1 week after treatment.

Example 9 platelet suspension for treatment of alopecia

2. The suspension can be used for treating alopecia.

3. The treatment mode comprises direct multipoint injection (dermis layer, subcutaneous shallow layer) at the alopecia position, smearing and combined use, wherein according to different application directions, 1-2 months of treatment can be referred to for 1 time, and 3-4 times of treatment can be referred to for each treatment course.

4. Treatment evaluation criteria 1) average hair density, 2) hair diameter and strength, 3) auxiliary evaluation criteria, scalp epidermis thickness and hair follicle number.

5. Conclusion of treatment:

After treatment with the iPSC-derived platelet rich suspension, the hair density of patients with androgenic alopecia (androgenetic alopecia, AGA) increased significantly, the percentage increase correlated significantly with the frequency of treatment, the hair diameter increased and the pullout test decreased, demonstrating a clear improvement in hair strength, and in addition, histopathological evaluation showed a significant increase in scalp epidermis thickness and hair follicle number after injection of the iPSC-derived platelet rich suspension.

The foregoing description is only of the preferred embodiments of the application and is not intended to limit the application thereto. All documents mentioned in this application are incorporated by reference in their entirety. Further, it will be understood that after reading the above teachings of the present application, those skilled in the art may make various changes or modifications to the present application without departing from the spirit and principles of the application, and such equivalent modifications are within the scope of the application as defined in the appended claims.

Claims

1. A composition for use in the treatment of arthritis comprising the following ingredients:

(2) Additive, 0.9% NaCl physiological saline and 5% human recombinant serum albumin;

wherein the composition is administered at a dose of 5mL.

2. The composition for treating arthritis according to claim 1, wherein said supplement further comprises 100U/mL thrombin lyophilized powder, 1% calcium gluconate and 0.05% type i collagenase.

3. A composition for use in the treatment of arthritis comprising the following ingredients:

(2) The additive comprises 10 mu MADP, 10-100 mu g/mL fibrinogen, 2mM CaCl ₂ and 0.05-0.5% type I collagenase.

4. A composition for the treatment of arthritis according to claims 1-3, wherein the platelet concentrate concentration is 5-20 x 10 ¹¹/L.

5. A composition for the treatment of arthritis according to claims 1-3, wherein the composition is administered 1 time per week, 3 times per course of treatment.

6. Use of a composition according to claims 1-3 for the preparation of a medicament for the treatment of arthritis.