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

Abstract

The invention provides a composition for treating arthritis, which comprises a blood platelet concentrated solution prepared by differentiation of human pluripotent induced stem cells (iPSC), and an additive for preparing a blood platelet suspension or gel. The invention aims to provide a functional platelet-rich composition which can be prepared in a large scale, is stable in batch and can be produced in a standardized manner in the aspect of treating arthritis. According to the composition, the platelet concentrate and platelet-rich plasma (PRP) separated from blood in the composition are completely different in component, the component is stable, and the slow-release growth factor is beneficial to tissue repair.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and particularly relates to the application of in vitro preparation of platelet-rich materials from induced pluripotent stem cells (iPSCs) in osteoarthritis. Background Art

[0002] There are more than 100 different types of arthritis, with different causes and treatment regimens. Among them, osteoarthritis (OA) and rheumatoid arthritis (RA) are the two most common types of arthritis. Osteoarthritis is a degenerative disease caused by multiple factors that lead to fibrosis, cracking, ulceration, and loss of articular cartilage, with joint pain as the main symptom. Approximately 240 million people worldwide are affected by it, and 50% of people over 65 years old are patients with osteoarthritis. Rheumatoid arthritis is a systemic autoimmune and multi-factor-related complex disease characterized by erosive arthritis. The global prevalence of rheumatoid arthritis is approximately 1%, and the male-to-female ratio is about 1:3.

[0003] The main pathological features of osteoarthritis are degradation and degeneration of articular cartilage, subchondral bone remodeling, synovial membrane and bone spur hyperplasia, etc. The pathogenesis of OA is complex, and cartilage degeneration involves cross-regulation between multiple pathways and phenotypes, such as the MAPK pathway, NF-κB pathway, apoptosis, autophagy, anabolism, and catabolism, etc., so it is extremely difficult to cure. The main pathological feature of rheumatoid arthritis is synovial inflammation of joints, and the formation of immune cell infiltration and pannus leads to persistent damage of cartilage and subchondral bone.

[0004] 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 the production of hyaluronic acid, and promote the formation of new blood vessels in joints, thereby increasing the amount of nutrients reaching the damaged area of articular cartilage. Research shows that platelet-derived growth factors IGF-1 (insulin-like growth factor 1) and TGF-β (transforming growth factor β) play key roles in chondrocyte differentiation, collagen synthesis, and matrix deposition. For example, in a rabbit knee osteoarthritis model, IGF-1 inhibits the NF-κB signaling pathway by regulating the MAPK and PI3K / Akt signaling pathways, and inhibits apoptosis by reducing the production of reactive oxygen species (ROS), thereby protecting chondrocytes. In addition, mouse studies have shown that the TGF-β / TAK1-FoxO1 cascade signaling pathway can regulate autophagy and homeostasis maintenance in articular cartilage.

[0005] Although platelet-rich plasma (PRP) is currently applied to numerous clinical treatment directions, such as orthopedics (bone injury repair, cartilage injury repair, etc.); sports injury (acute sports injury, chronic mechanical injury, etc.); general surgery (acute trauma, treatment of chronic non-healing wounds, etc.); burn and plastic surgery (burn wounds, skin tissue regeneration, skin and fat tissue liquefaction, etc.); and dentistry (periodontal disease, jaw surgery, etc.). However, since it is generally prepared by autologous blood sampling, it is often restricted and affected by the donor background and sampling volume. Moreover, due to the diversity of individual blood quality and preparation methods (there is currently no unified standard preparation process across regions), past data cannot provide extensive and effective guidance for clinical practice, so clinical practice relies more on the hospital system and doctor experience. In addition, since the preparation process of PRP is all carried out in vitro, it will also bring some operation-related risks such as infection.

[0006] The preparation of iPSC-platelets is a prerequisite for producing a uniform platelet concentrate product and also a prerequisite for generating corresponding treatment standards. The preparation of iPSC platelets is achieved through the in vitro differentiation of iPSCs, and the whole-process in vitro differentiation and regulation path is currently relatively clear. In 2018, the team of Koji Eto at Kyoto University in Japan published that using an 8L turbulent blood platelet reactor, 1×10 11 platelets can be produced at one time, and the production efficiency of 70 - 80 platelets per megakaryocyte is achieved, laying a foundation for the in vitro production of platelets.

[0007] However, in the treatment of arthritis, the existing technology still lacks functional platelet-rich plasma that can be prepared in large quantities, has stable quality in each batch, and can be produced standardized. Summary of the Invention

[0008] The present invention provides a method for in vitro differentiating human induced pluripotent stem cells (iPSCs) and generating platelets using a reactor and activating them in vitro, and finally provides a platelet composition for treating osteoarthritis or rheumatoid arthritis.

[0009] To achieve the above object, the present invention provides the following technical solutions:

[0010] The present invention provides a composition for treating arthritis, comprising the following components:

[0011] (1) A platelet concentrate prepared by differentiating pluripotent induced stem cells;

[0012] (2) Additives: normal saline of 0.9% NaCl and 5% human recombinant serum albumin; wherein, the administration dose of the aforementioned composition is 5 mL.

[0013] Preferably, the above-mentioned additive further comprises freeze-dried thrombin at 100 U / mL, calcium gluconate at 1%, and type I collagenase at 0.05%.

[0014] The present invention provides a composition for treating arthritis, which is characterized by comprising the following components:

[0015] (1) Platelet concentrate prepared by differentiating pluripotent induced stem cells;

[0016] (2) Additive: 10 μM ADP, 10 - 100 μg / mL fibrinogen, 2 mM CaCl 2 and 0.05 - 0.5% type I collagenase; wherein, the administration dose of the aforementioned composition is 5 mL.

[0017] Preferably, the concentration of the above-mentioned platelet concentrate is 5 - 20×10 11 / L.

[0018] Preferably, the application frequency of the above-mentioned composition and the above-mentioned gel is once a week, and every three times is a treatment course.

[0019] More preferably, the application of the above-mentioned composition in the preparation of a drug for treating arthritis.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] (1) The process of differentiating iPSC into hematopoietic progenitor stem cells is divided into three stages, and each stage has its corresponding culture medium formula, which helps to improve the purity of CD34 + cells.

[0022] (2) During the process of differentiating hematopoietic progenitor stem cells into megakaryocytes, adding 50 ng / mL IL-21, 5 nM Tazemetostat, 200 nM Eltrombopag, and 200 nM iBET151 to the culture medium can promote the differentiation and proliferation of megakaryocytes.

[0023] (3) After cryopreservation and resuscitation of megakaryocytes, and before using the reactor, adding 0.5 ng / mL collagen and 5 nM Fingolimod HCl helps to promote platelet maturation.

[0024] (4) The reactor for generating platelets has a high-density grid structure, and the function of the grid structure is to cut the liquid flow, which can generate intense megakaryocyte fragmentation and help release platelets.

[0025] (5) Compared with PRP separated from blood, the iPSC platelet preparation is relatively stable in composition, and the slow-release growth factors help tissue repair.

[0026] (6) The formulation methods (suspension formula, preparation formula for activated suspension, gel preparation formula and preparation method) have never been used in the application scenario of iPSC - platelets.

[0027] (7) It can efficiently produce platelet - derived growth factor, and its properties are stable in batches, which can produce effective therapeutic effects and generate reference clinical data for arthritis treatment. Brief Description of the Drawings

[0028] The specification drawings forming a part of this application are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not unduly limit the present invention. In the drawings:

[0029] Figure 1 are the morphology (a) of hematopoietic progenitor cells (Hematopoietic progenitor cells) generated from induced pluripotent stem cells (iPSC), the cell size (b) of hematopoietic stem cells, and CD34 obtained through passage purification + cells (c) and their purity (d). CD34: A marker mainly expressed in hematopoietic stem cells and hematopoietic progenitor cells. BF: brightfield, bright - field view.

[0030] Figure 2 are the data comparisons of iPSC - derived megakaryocytes (iMKs) from a newborn male (A) and a 34 - year - old female (B) after cryopreservation and resuscitation, including morphology (b, c), proliferation ability (d, e), cell viability (f) and cell size (g).

[0031] Figure 3 are the in vitro maturation and shedding process of platelets (a), and the proportion (b), overall size (c) of platelets produced by a single megakaryocyte in static and reactor environments; through morphological observation, it is found that intense megakaryocyte fragmentation and platelet release can occur in the reactor within 72 hours (d). Through flow cytometry detection, it is found that iPSC - derived platelets (iPLTs) have similar marker expression (f) compared with platelets derived from peripheral blood (e). BF: bright field, bright - field view. PB - PLTS: Platelets derived from peripheral blood.

[0032] Figure 4 are the states of unactivated and activated platelet suspensions during the preparation process of platelet suspensions. Detailed Description of the Invention

[0033] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments. The protection scope of the present invention is not limited to the following embodiments. It should also be understood that the terms used in the embodiments of the present invention are for the purpose of describing specific implementation manners and not for limiting the protection scope of the present invention, and are not uniquely defined. Without departing from the spirit and scope of the inventive concept, changes and advantages that can be conceived by those skilled in the art are included in the present invention, and the appended claims and any equivalents thereof are the protection scope of the present invention.

[0034] All technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. In other cases, certain terms used herein will be clarified in the specification. The experimental methods without specific conditions noted in the following examples are the general knowledge and common sense of those skilled in the art. The embodiments in the present application and the features in the embodiments can be combined with each other.

[0035] The characteristics and advantages of the present invention can be further understood through the following detailed description in conjunction with the accompanying drawings. The provided embodiments are only illustrative of the methods of the present invention and do not limit the rest of the content disclosed by the present invention in any way.

[0036] In order to provide a platelet preparation with stable components, as well as a preparation method that can be mass-produced, batch-stable, and standardized, the present invention utilizes the iPSC platelet preparation method, reactor, suspension formulation, activated suspension formulation, gel formulation, and preparation method to achieve technical effects.

[0037] The present invention provides a method for preparing platelets using stem cells. First, the process of differentiating induced pluripotent stem cells (iPSC) into hematopoietic progenitor stem cells (HPC) is divided into three stages, and each stage has its own corresponding culture medium formulation:

[0038] Table 1.

[0039]

[0040] Secondly, during the process of differentiating HPC into megakaryocytes (iMK), adding 50 ng / mL IL-21, 5 nM Tazemetostat, 200 nM Eltrombopag, and 200 nM iBET151 to the culture medium can promote the differentiation and proliferation of megakaryocytes.

[0041] Finally, after cryopreservation and resuscitation of megakaryocytes and before using the reactor, add a maturation process from static to dynamic (see points 1 and 2 in Example 3), and add 0.5 ng / mL Collagen and 5 nM Fingolimod HCl to promote platelet maturation.

[0042] The present invention uses a reactor to generate platelets. In this reactor, there is a high-density grid structure, and the function of the grid structure is to cut the liquid flow. There are two ways for the grid structure to cut the liquid flow: 1) the grid is static and the liquid flow is dynamic; or 2) the grid is dynamic and the liquid flow is static.

[0043] Although the method for preparing iPSC platelet preparations in the present invention refers to the traditional PRP preparation idea, the iPSC platelet preparations and the PRP separated from blood are completely different in composition, and the composition of the PRP separated from blood is relatively unstable. Traditional iPSC platelets include a large proportion of plasma components. Due to different individual / blood bank sources, their components and proportions vary greatly, which can lead to differences in effects during application; the components of iPSC-platelets are determined, the batches are stable, there are no component differences caused by different sampling individuals, and they can be prepared on a large scale and strictly quality controlled, so as to obtain a standardized product with strict control over the production process and product quality. Therefore, it can play a good guiding and reference role for the dosage and effect of clinical applications. Correspondingly, the platelet-rich suspension product derived from iPSC is different from peripheral blood PRP and does not contain white blood cell components and plasma components. The platelet-rich suspension derived from iPSC contains iPSC-induced platelets and 5% albumin components. The platelet growth factors produced in large quantities after activation can play a role in repairing tissue damage. Especially for the gel-like PRP, due to the fiber intertwining and winding of components such as platelets and growth factors, it can play a slow-release effect, so as to achieve a relatively long-term use effect and also has a role in tissue repair.

[0044] The formula of the suspension and gel additives of the present invention is as follows:

[0045] Table 2.

[0046]

[0047]

[0048] In addition, the iPSC platelet preparations of the present invention can be applied to different scenarios, such as treating arthritis, tennis elbow, promoting wound healing, treating hair loss, etc.; they can also be applied to the beauty field.

[0049] Example 1: Differentiate iPSC into hematopoietic progenitor cells (iHPCs)

[0050] The iPSC of the present invention is sampled from hospital skin biopsies, and the primary fibroblasts are cultured in the laboratory and screened to obtain an ideal iPSC cell line after reprogramming.

[0051] 1. Dissociate iPSCs into single cells and evenly seed them in a culture dish pre-coated with human recombinant laminin (rhLaminin-521, Thermofisher). The culture medium is Essential 8 (Thermofisher) supplemented with 30 ng / mL VEGF-A165, 10 ng / mL bFGF, 5 μM Y-27632, 2 mM Chir-99021, and 20 ng / mL Activin A, and the culture time is 2 days.

[0052] 2. Then change the culture medium to HDM, with the following formulation:

[0053] HDM medium: Add 1x ITS, 1x glutamax, 0.45 mM monothioglycerol, 50 μg / mL ascorbic acid, and 20% KO-SRM to Iscove modified Dulbecco (IMDM).

[0054] Subsequently, add 30 ng / mL VEGF-A165, 10 ng / mL bFGF, 20 ng / mL BMP4, 5 μM Y-27632, and 20 ng / mL Activin A, and continue to culture for 4 days.

[0055] 3. Then change the culture medium to HDM medium and add 30 ng / mL VEGF-A165, 10 ng / mL bFGF, 50 ng / mL stem cell factor (SCF), 50 ng / mL thrombopoietin (TPO), 2 U / mL erythropoietin (EPO), and 5 μM Y-27632. Optionally, further add 50 ng / mL IL-3 and 50 ng / mL IL-6, and continue to culture for 8 days.

[0056] 4. Collect the hematopoietic precursor stem cells (iHPCs) growing in suspension, collect the culture medium supernatant containing iHPCs, centrifuge at 1000 rpm for 5 minutes.

[0057] For the results, see Figure 1 . The hematopoietic precursor stem cells (HPC) generated from iPSCs in this example Figure 1 a) have a cell size similar to that of CD34 cells isolated from peripheral blood + ( Figure 1 b), and through passage purification, the obtained CD34 + cells Figure 1 c) can reach a purity of about 90% Figure 1d), More starting materials help reduce interference in subsequent differentiation processes and do not generate excessive miscellaneous cells.

[0058] Example 2: Differentiation and Proliferation of Megakaryocytes

[0059] I. Differentiate hematopoietic precursor stem cells (iHPC) into megakaryocytes (iMKs)

[0060] 1. Collect the hematopoietic precursor stem cells obtained in Example 1 and seed them in a culture dish pre-coated with 0.1% gelatin. The culture medium is HDM medium supplemented with 20 ng / mL VEGF-A165, 5 ng / mL bFGF, 50 ng / mL thrombopoietin (TPO), 20 ng / mL IL-3, 20 ng / mL IL-6, 50 ng / mL IL-21, 5 nM Tazemetostat, 200 nM Eltrombopag, and 200 nM iBET151, and continue culturing for 15 - 20 days.

[0061] 2. Collect the suspended megakaryocytes.

[0062] II. In vitro Proliferation of Megakaryocytes (iMKs)

[0063] 1. Transfer the megakaryocytes obtained in the previous step I. to a low-attachment culture flask / shaker flask, change the culture medium to HDM supplemented with 50 ng / mL thrombopoietin (TPO), 50 ng / mL stem cell factor (SCF), 50 ng / mL IL-21, and 5 μM Y-27632, and place it on a horizontal shaking incubator for amplification, with the rotation speed adjusted to 120 - 150 rpm.

[0064] 2. Megakaryocytes can be amplified in vitro for several months while maintaining their properties unchanged. The passage cycle is 3 days, and the seeding density for each passage is 5×10 6 / mL.

[0065] 3. Megakaryocytes can be cryopreserved. The cryopreservation solution is HDM + 5% BSA + 5% DMSO, and after programmed freezing, they are stored in liquid nitrogen.

[0066] The results are shown in Figure 2 . Two different background iPSCs Figure 2 a) - derived megakaryocytes (iMKs) from a newborn male (A) and a 34-year-old female (B), after cryopreservation and recovery, can maintain a dispersed and uniform morphology during long-term dynamic proliferation culture Figure 2 b, Figure 2 c), a proliferation ability of 10,000-fold in 15 days Figure 2 d, Figure 2 e), and a relatively stable cell viability Figure 2f) and cell size ( Figure 2 g).

[0067] Example 3: Production of Platelets

[0068] 1. Suspend the megakaryocytes obtained in Example 2 in PM medium, add 50 ng / mL thrombopoietin (TPO) and 5 μM Y-27632, and statically culture for 1 day.

[0069] PM medium: Add 1x ITS, 1x glutamax, 0.45 mM monothioglycerol, 50 μg / mL ascorbic acid, 10 U heparin, and 5% human plasma to Iscove modified Dulbecco (IMDM).

[0070] 2. Replace the medium with PM medium, add 50 ng / mL thrombopoietin (TPO), 0.5 ng / mL collagen, 5 nM Fingolimod HCl and 5 μM Y-27632, statically culture for 1 day, and then continue to culture on a horizontal shaking incubator for 1 day, with the rotation speed adjusted to 120 - 150 rpm.

[0071] 3. Transfer the megakaryocytes obtained in the above steps to a bioreactor for platelet maturation in a 1 L system, and continue to culture for 5 - 6 days. The culture environment is 37°C, 5% CO 2 , and the maximum horizontal liquid streamline velocity in the reactor is 30 cm / s.

[0072] The specifications of the bioreactor are as follows: The appearance is a cylindrical structure, the height of the external tank is 160 mm, the bottom diameter is 130 mm, and the thickness of the culture container is 5 mm; it is embedded with a multi-layer movable grid structure, with a variable-direction rotational movement mode, a rotational speed of 120 - 150 rpm, and a maximum liquid flow linear velocity of 30 cm / s.

[0073] The results are shown in Figure 3 . The in vitro maturation and shedding process of platelets in this example ( Figure 3 a), in the parallel control of the static environment and the reactor environment, the proportion of platelets produced by a single megakaryocyte under reactor conditions can reach more than 1:100 ( Figure 3 b), and the overall size is closer to platelets from peripheral blood ( Figure 3 c), through morphological observation, it is found that intense megakaryocyte fragmentation and platelet release can occur in the reactor within 72 hours ( Figure 3d). By flow cytometry, it was found that iPSC-derived platelets (iPLTs) had similar marker expressions Figure 3 compared with platelets Figure 3 from peripheral blood

[0074] Example 4: Preparation methods of platelet suspension and gel

[0075] 1. Human iPSC-derived platelet-rich suspension

[0076] After concentrating and purifying the iPSC platelets in Example 3, they were suspended in physiological saline containing 0.9% NaCl at a concentration of 5 - 20×10 11 / L, and 5% human recombinant serum albumin (rhHSA) was added.

[0077] 2. Activated human iPSC-derived platelet-rich suspension

[0078] Based on the suspension in point 1 above, 100 U / mL freeze-dried thrombin, 1% calcium gluconate, and 0.05% type I collagenase were added.

[0079] 3. Human iPSC-derived platelet-rich gel

[0080] The iPSC platelets obtained in Example 3 were concentrated to 1×10 11 / L or more, suspended in PBS, and 10 μM ADP, 10 - 100 μg / mL fibrinogen, 2 mM CaCl 2 and 0.05 - 0.5% type I collagenase were added to the solution. It was placed in a sterile environment at room temperature and allowed to crosslink for 5 minutes. Then it was centrifuged at 200 rpm for 15 minutes, and then continued to be centrifuged at 1500 rpm for 2 minutes. The supernatant was discarded, and the lower layer rich in activated platelet gel could be obtained.

[0081] The results are shown in Figure 4 . During the preparation of the platelet suspension, the unactivated platelet suspension (described in method 1 of this example) showed a relatively clear state. After activation (described in method 2 of this example), due to the aggregation of platelets and the entanglement of fibrin, the suspension immediately became turbid (the control group was platelets isolated from peripheral blood). Among them, compared with the platelets isolated from peripheral blood in the control group, the aggregation effect of iPSC platelets was better.

[0082] Example 5: Application of platelet suspension in the treatment of arthritis

[0083] 1. The preparation method of human iPSC-derived platelet-rich suspension was as described in Examples 1 - 4.

[0084] 2. The above platelet suspension was used to treat arthritis, specifically including osteoarthritis and rheumatoid arthritis.

[0085] 3. The injection method is: intra-articular injection of the knee joint under ultrasound guidance, using a 22-gauge needle, 5 mL of fresh human iPSC-derived platelet-rich suspension each time. After injection, the knee joint is flexed and extended passively 5 times, and the patient rests for 10 minutes. Inject once a week, 3 times per course of treatment. The patients are followed up within 12 months after injection.

[0086] 4. The evaluation methods for treatment improvement are as follows: 1) The average knee joint pain score (11-point numerical rating scale, 0 = no pain - 10 = the most severe pain); 2) Measuring the volume of the medial tibial cartilage by MRI; 3) Evaluating pain, function, quality of life, overall changes, and joint structure, etc. using 31 auxiliary evaluation indicators (25 symptom-related and 6 MRI evaluations).

[0087] Example 6: Application of platelet suspension in the treatment of tennis elbow

[0088] 1. The preparation method of human iPSC-derived platelet-rich suspension is as in Examples 1 to 4.

[0089] 2. The above platelet suspension is used to treat tennis elbow.

[0090] 3. The treatment method is: block the injection site with 0.5% bupivacaine and adrenaline, and then inject 2 - 3 mL of the prepared human iPSC-derived platelet-rich suspension into the extensor carpi radialis brevis tendon and the surrounding area by tendon puncture. The needle is inserted once without being withdrawn and the direction is changed 5 times to inject the tendon for infiltration.

[0091] 4. The evaluation methods for treatment improvement are as follows: 1) At 4, 8, 12, 16, 20, and 24 weeks after treatment, if the pain score improvement of the VAS pain score for wrist extension against resistance (evaluating the severity of pain by the visual analogue method, VASRWE) is 25% or more compared with the baseline, it is considered a success; 2) Auxiliary evaluation indicators: The patient-rated tennis elbow evaluation questionnaire (PRTEE); Extended wrist examination.

[0092] Example 7: Application of platelet suspension in wound healing treatment

[0093] 1. The preparation method of human iPSC-derived platelet-rich suspension is as in Examples 1 to 4.

[0094] 2. The above suspension is used to promote wound healing, such as in the healing of trauma and burns.

[0095] 3. The treatment method is: after surgical treatment of the trauma, before closing and suturing the incision, apply the iPSC-derived platelet-rich suspension into the wound, at least 5 mL at each site, and the platelet content is not less than 1×10 6 / μL, and the dosage is adjusted according to the wound area and the degree of injury.

[0096] 4. The evaluation indicators for treatment improvement are: 1) wound healing time; 2) wound infection assessment; 3) wound healing quality.

[0097] For general wounds, unactivated platelet PRP is used to enable the slow release of growth factors to promote healing. For severely injured or infection-prone wounds, activated platelets are used to enrich and rapidly release a large amount of growth factors at the wound site in a short time, thereby rapidly promoting wound healing.

[0098] Example 8: Application of platelet suspension in the field of cosmetology

[0099] 1. The preparation method of human iPSC-derived platelet-rich suspension is as described in Examples 1 to 4.

[0100] 2. The above suspension is used in the field of cosmetology.

[0101] 3. The treatment methods are: direct injection (dermis layer, superficial subcutaneous layer), application, and combined use. According to different application directions, it can be referred to that the treatment is carried out once every 1 to 2 months, and 3 to 4 times for each course of treatment.

[0102] 4. Precautions for treatment:

[0103] The treatment with human iPSC-derived platelet-rich suspension does not require a recovery period, and normal work can be resumed the day after treatment. Swelling or bleeding points may occur in the treatment area after treatment. Generally, mild swelling can subside on the same day, and bleeding points can generally disappear in 2 to 3 days. It should be noted that:

[0104] (1) Women should prepare and use concentrated platelet products during non-menstrual periods. Pregnant, lactating, and women planning to conceive, as well as patients with anemia, abnormal blood coagulation function, and abnormal liver function, are prohibited from using;

[0105] (2) Keep the treatment area dry for 24 hours after treatment, do not get it wet, do not use other irritating skin care products; pay attention to sun protection, do not take anticoagulant drugs 2 weeks before treatment; it is not recommended to take a steam bath or massage 1 week after treatment.

[0106] Example 9: Application of platelet suspension in the treatment of hair loss

[0107] 1. The preparation method of human iPSC-derived platelet-rich suspension is as described in Examples 1 to 4.

[0108] 2. The above suspension is used for the treatment of hair loss.

[0109] 3. The treatment methods are: direct multi-point injection (dermis layer, superficial subcutaneous layer), application, and combined use at the hair loss position. According to different application directions, it can be referred to that the treatment is carried out once every 1 to 2 months, and 3 to 4 times for each course of treatment.

[0110] 4. Treatment evaluation criteria: 1) Average hair density; 2) Hair diameter and strength; 3) Auxiliary evaluation indicators: Epidermal thickness and hair follicle number of the scalp.

[0111] 5. Treatment conclusion:

[0112] In patients with androgenetic alopecia (AGA), after treatment with iPSC-derived platelet-rich suspension, the hair density increased significantly during follow-up, and the percentage increase was significantly correlated with the treatment frequency; the hair diameter increased and the results of the hair pull test decreased, indicating a clear improvement in hair strength; in addition, histopathological evaluation showed that after injection of iPSC-derived platelet-rich suspension, the epidermal thickness and hair follicle number of the scalp increased significantly.

[0113] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. All documents mentioned in the present invention are hereby incorporated by reference in their entirety in this application. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention within the spirit and principle of the present invention, and these equivalent forms of modification also fall within the scope defined by the claims of this application.

Claims

1. A composition for treating arthritis, characterized in that, it comprises the following components: (1) Platelet concentrate prepared by differentiating pluripotent induced stem cells; (2) Additives: normal saline of 0.9% NaCl and 5% human recombinant serum albumin; wherein, the administration dose of the composition is 5 mL.

2. The composition for treating arthritis according to claim 1, characterized in that, the additive further comprises 100 U / mL freeze-dried thrombin, 1% calcium gluconate and 0.05% type I collagenase.

3. A composition for treating arthritis, characterized in that, it comprises the following components: (1) Platelet concentrate prepared by differentiating pluripotent induced stem cells; (2) Additives: 10 μM ADP, 10 - 100 μg / mL fibrinogen, 2 mM CaCl 2 and 0.05 - 0.5% type I collagenase.

4. The composition for treating arthritis according to claims 1 to 3, characterized in that, The concentration of the platelet concentrate is 5 to 20×10 11 / L.

5. The composition for treating arthritis according to claims 1 to 3, characterized in that, the administration frequency of the composition is once a week, and every three times is a treatment course.

6. Use of the composition according to claims 1 to 3 in the preparation of a drug for treating arthritis.

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