Application of human iPSC (induced pluripotent stem cell)-derived platelet-rich suspension in treatment of alopecia

Abstract

The invention provides a composition for treating alopecia, 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 alopecia. 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 relates to the application of in vitro preparation of platelet-rich materials from induced pluripotent stem cells (iPSCs) in the treatment of alopecia, specifically to the application direction in the treatment of androgenetic alopecia (AGA). Background Art

[0002] Alopecia is a common disease that can affect most people, especially men. The most common type is androgenetic alopecia (AGA). There are many reasons for alopecia. One explanation is that chronic micro-inflammation around the hair follicles will exacerbate the expression of pro-inflammatory cytokines, leading to oxidative stress responses. This oxidative stress, together with high levels of androgens, genetic factors, and environmental factors (such as stress), affects the release of adrenocorticotropic hormone and cortisol levels, thus causing alopecia. Since the disease does not have a single pathogenesis, different treatment methods targeting different pathogenesis need to be combined simultaneously for effective treatment.

[0003] The reconstruction of healthy hair follicle cells is affected by various growth factors, which can stimulate hair to enter the growth phase, facilitate cell proliferation, improve the extracellular matrix, and promote blood circulation reconstruction. In addition to classic AGA therapies such as minoxidil or finasteride, several new treatment strategies have been proposed in recent years, providing multiple options for individualized treatment of patients. Among these new therapies, the efficacy of platelet-rich plasma (PRP) has been verified in some randomized controlled trials.

[0004] PRP treatment has been widely used in dermatology and plastic surgery, and has been successfully applied to the treatment of facial rejuvenation, wrinkles, atrophic acne scars, cosmetic surgery, periorbital rejuvenation, dermal filling, etc. Since the growth factors contained in PRP play an important role in skin regeneration, fibroblast and macrophage chemotaxis, fibroblast proliferation, and extracellular matrix synthesis, this therapy is expected to have a beneficial effect on hair follicle regeneration, especially in the treatment of AGA.

[0005] Scientific research has found that the proportion of various growth factors contained in PRP is similar to the normal level in the body, while the concentration is 5 to 10 times the normal value in the body. PRP is a treasure trove of a series of special protein growth factors, including platelet-derived growth factor (PDGF), vascular endothelial growth factor (VEGF), transforming growth factor β (TGF-β), epidermal growth factor (EGF), fibroblast growth factor (FGF), brain-derived neurotrophic factor (BDNF), etc. Growth factors, as the name implies, are the starters of cell factories. Through their coordinated cooperation, they promote the proliferation of various types of tissue cells in the body, stimulate epithelial regeneration and the formation of new blood vessels. In addition, the fibrous network structure formed by a large amount of fibrinogen contained in PRP can provide a good scaffold for repair cells and stimulate soft tissue regeneration. Injecting PRP into the scalp at the hair loss site, the released growth factors will cooperate with each other to stimulate the proliferation of dermal papilla cells that control hair follicle growth, thereby promoting hair structure formation and the differentiation of hair follicle stem cells. PRP not only prolongs the growth phase of hair, prevents hair follicle atrophy and disappearance, but also makes hair follicles that are already in the resting phase return to the growth phase.

[0006] However, in the treatment of hair loss, the existing technology still lacks functional platelet-rich preparations that can be mass-produced, have stable quality in each batch, and can be produced in a standardized manner. Summary of the Invention

[0007] The present invention provides a method for differentiating human pluripotent induced stem cells (iPSC, induced pluripotent stem cell) in vitro, generating platelets using a reactor, and activating them in vitro, and finally provides a platelet composition for treating hair loss.

[0008] To achieve the above object, the present invention provides the following technical solutions:

[0009] The present invention provides a composition for treating hair loss, comprising the following components:

[0010] (1) Platelet concentrate prepared by differentiating pluripotent induced stem cells;

[0011] (2) Additives: normal saline of 0.9% NaCl and 5% human recombinant serum albumin; wherein, the aforementioned composition is applied to the hair loss position of the dermal layer and the shallow subcutaneous layer by direct multi-point injection, smearing, and / or combined use of injection and smearing.

[0012] Preferably, the above additives further comprise freeze-dried thrombin powder of 100 U / mL, 1% calcium gluconate, and 0.05% type I collagenase.

[0013] The present invention also provides a composition for treating hair loss, comprising the following components:

[0014] (1) Platelet concentrate prepared by differentiating pluripotent induced stem cells;

[0015] (2) Additives: 10 μM ADP, 10 - 100 μg / mL fibrinogen, 2 mM CaCl 2 and 0.05 - 0.5% type I collagenase; wherein, the aforementioned composition is directly administered by multi - point injection, smearing, and / or combined use of injection and smearing at the alopecia positions in the dermal layer and the superficial subcutaneous layer.

[0016] Preferably, the concentration of the above - mentioned platelet concentrate is 5 - 20×10 11 / L.

[0017] Preferably, the above - mentioned composition is administered once every 1 - 2 months, and every 3 - 4 times is a treatment course.

[0018] More preferably, the application of the above - mentioned composition in the preparation of a drug for treating alopecia.

[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0020] (1) The process of differentiating iPSCs into hematopoietic precursor 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.

[0021] (2) During the process of differentiating hematopoietic precursor stem cells into megakaryocytes, 50 ng / mL IL - 21, 5 nM Tazemetostat, 200 nM

[0022] Eltrombopag and 200 nM iBET151 are added to the culture medium, which can promote the differentiation and proliferation of megakaryocytes.

[0023] (3) After cryopreservation and resuscitation of megakaryocytes, and before using the reactor, adding

[0024] 0.5 ng / mL collagen and 5 nM Fingolimod HCl helps to promote platelet maturation.

[0025] (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 cause intense megakaryocyte fragmentation and help release platelets.

[0026] (5) Compared with PRP separated from blood, the iPSC platelet preparation is more stable in composition, and the slow - release growth factors help tissue repair.

[0027] (6) The formulation methods (suspension formula, preparation formula of activated suspension, gel preparation formula and preparation method) have never been used in the application scenario of iPSC-platelets.

[0028] (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 hair loss treatment. Brief Description of the Drawings

[0029] 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:

[0030] Figure 1 They 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 of view.

[0031] Figure 2 They are the comparison of various data of induced pluripotent stem cell (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 survival rate (f) and cell size (g).

[0032] Figure 3 They 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 of view. PB-PLTS: Platelets derived from peripheral blood.

[0033] Figure 4 They are the states of unactivated and activated platelet suspensions during the preparation process of platelet suspensions. Detailed Description of the Invention

[0034] 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 embodiments, rather than 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.

[0035] 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 pertains. In other cases, certain terms used herein will be clarified in the specification. The experimental methods without specific conditions noted in the following embodiments are the general knowledge and common general knowledge of those skilled in the art. The embodiments in this application and the features in the embodiments can be combined with each other.

[0036] 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 remaining content disclosed by the present invention in any way.

[0037] 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.

[0038] 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 corresponding culture medium formulation:

[0039] Table 1.

[0040]

[0041]

[0042] 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.

[0043] Finally, before cryopreserving and thawing megakaryocytes and before using the reactor, add a maturation process that transforms from static to dynamic (see points 1 and 2 of Example 3), and add 0.5 ng / mL Collagen and 5 nM Fingolimod HCl to promote platelet maturation.

[0044] The present invention uses a reactor to generate platelets. Among them, the reactor has 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.

[0045] Although the method for preparing iPSC platelet preparations in the present invention refers to the traditional PRP preparation idea, the iPSC platelet preparations and PRP separated from blood are completely different in composition, and the composition of 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 in the dosage and effect of clinical applications. Correspondingly, for example: The platelet-rich suspension product derived from iPSC is different from peripheral blood PRP, and it 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 intertwined with platelets and growth factor components, 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.

[0046] The formulation of the suspension and gel additives of the present invention is as follows:

[0047] Table 2.

[0048]

[0049] 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.

[0050] Example 1: Differentiate iPSC into hematopoietic progenitor cells (iHPCs)

[0051] The iPSCs of the present invention are obtained from skin biopsies in the hospital, and the primary fibroblasts are cultured in the laboratory and reprogrammed, and then an ideal iPSC cell line is screened.

[0052] 1. Dissociate the 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.

[0053] 2. Then change the culture medium to HDM, and the formula is as follows:

[0054] HDM culture medium: Add 1x ITS, 1x glutamax, 0.45 mM monothioglycerol, 50 μg / mL ascorbic acid, and 20% KO-SRM to Iscove modified Dulbecco (IMDM).

[0055] Thereafter, 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.

[0056] 3. Then change the culture medium to HDM culture 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, 50 ng / mL IL-3 and 50 ng / mL IL-6 can be further added, and continue to culture for 8 days.

[0057] 4. Collect the hematopoietic precursor stem cells (iHPCs) growing in suspension, collect the culture medium supernatant containing iHPCs, and centrifuge at 1000 rpm for 5 minutes.

[0058] The results are shown in Figure 1 . The hematopoietic precursor stem cells (HPC) generated from the iPSCs in this example ( Figure 1 a) have a cell size similar to that of CD34 + cells isolated from peripheral blood ( Figure 1b), and through passage purification, the obtained CD34 + cells ( Figure 1 c) The purity can reach about 90% ( Figure 1 d), more starting materials help reduce interference in the subsequent differentiation process and do not generate excessive heterologous cells.

[0059] Example 2: Differentiation and proliferation of megakaryocytes

[0060] I. Differentiate hematopoietic precursor stem cells (iHPC) into megakaryocytes (iMKs)

[0061] 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.

[0062] 2. Collect the suspended megakaryocytes.

[0063] II. In vitro proliferation of megakaryocytes (iMKs)

[0064] 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, and adjust the rotation speed to 120 - 150 rpm.

[0065] 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 each time is 5×10 6 / mL.

[0066] 3. Megakaryocytes can be cryopreserved. The cryopreservation solution is HDM + 5% BSA + 5% DMSO, and after programmed freezing, it is stored in liquid nitrogen.

[0067] The results are shown in Figure 2 . Two different background iPSC-derived Figure 2 a) megakaryocytes (iMKs) from a newborn male (A) and a 34-year-old female (B), after cryopreservation and resuscitation, can maintain a dispersed and uniform morphology during long-term dynamic proliferation culture ( Figure 2 b, Figure 2c), A proliferation ability of a million times in 15 days Figure 2 d、 Figure 2 e), And relatively stable cell viability Figure 2 f) and cell size Figure 2 g).

[0068] Example 3: Production of platelets

[0069] 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.

[0070] 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).

[0071] 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.

[0072] 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.

[0073] The reactor specifications 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.

[0074] 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 3c), Through morphological observation, it was found that severe megakaryocyte fragmentation and platelet release could occur in the reactor within 72 hours. Figure 3 d). Through flow cytometry detection, it was found that iPSC-derived platelets (iPLTs) and platelets derived from peripheral blood Figure 3 e) had similar marker expression Figure 3 f).

[0075] Example 4: Preparation methods of platelet suspension and gel

[0076] 1. Human iPSC-derived platelet-rich suspension

[0077] 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.

[0078] 2. Activated human iPSC-derived platelet-rich suspension

[0079] On the basis of the suspension in the above point 1, 100 U / mL freeze-dried thrombin, 1% calcium gluconate and 0.05% type I collagenase were added.

[0080] 3. Human iPSC-derived platelet-rich gel

[0081] The iPSC platelets obtained in Example 3 were concentrated to 1×10 11 / L or more, suspended with PBS, 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, placed in a sterile environment at room temperature, and allowed to stand and crosslink for 5 minutes. Centrifuged at 200 rpm for 15 minutes, and then continued to centrifuge at 1500 rpm for 2 minutes, and the supernatant was discarded to obtain the lower layer rich in activated platelet gel.

[0082] The results are shown in Figure 4 . During the preparation process 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 platelet aggregation and fibrin entanglement, 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.

[0083] Example 5: Application of platelet suspension in the treatment of arthritis

[0084] 1. The preparation method of human iPSC-derived platelet-rich suspension is as described in Examples 1 - 4.

[0085] 2. Use the above platelet suspension for treating arthritis, specifically including osteoarthritis and rheumatoid arthritis.

[0086] 3. The injection method is: intra-articular injection into 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, and follow up the patient within 12 months after injection.

[0087] 4. The evaluation methods for treatment improvement are: 1) average knee joint pain score (11-point numerical rating scale, 0 = no pain - 10 = 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 secondary evaluation indicators (25 symptom-related and 6 MRI evaluations).

[0088] Example 6: Application of platelet suspension in the treatment of tennis elbow

[0089] 1. The preparation method of the human iPSC-derived platelet-rich suspension is as described in Examples 1 to 4.

[0090] 2. Use the above platelet suspension for treating tennis elbow.

[0091] 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 needling. The needle is inserted once without being withdrawn and the direction is changed 5 times to inject the tendon for infiltration.

[0092] 4. The evaluation methods for treatment improvement are: 1) at 4, 8, 12, 16, 20, and 24 weeks after treatment, if the pain score improvement of the VAS for wrist extension against resistance (evaluating the severity of pain using the visual analogue method, VASRWE) compared with the baseline is 25% or more, it is considered a success; 2) secondary evaluation indicators: patient-rated tennis elbow evaluation questionnaire (PRTEE); extended wrist examination.

[0093] Example 7: Application of platelet suspension in the treatment of wound healing

[0094] 1. The preparation method of the human iPSC-derived platelet-rich suspension is as described in Examples 1 to 4.

[0095] 2. Use the above suspension to promote wound healing, such as in the healing of trauma and burns.

[0096] 3. The treatment method is as follows: after the trauma is surgically treated, before closing and suturing the incision, the iPSC-derived platelet-rich suspension is applied into the wound, with at least 5 mL for each site, and the platelet content is not less than 1×10 6 / μL, and the dosage is adjusted according to the trauma area and the degree of injury.

[0097] 4. The evaluation indicators for treatment improvement are: 1) wound healing time; 2) wound infection assessment; 3) wound healing quality.

[0098] For general wounds, unactivated platelet PRP is used to promote healing by slowly releasing growth factors. For severely damaged or infection-prone traumas, activated platelets are used to enrich and rapidly release a large amount of growth factors at the trauma site in a short time, so as to rapidly promote injury healing.

[0099] Example 8: Application of platelet suspension in the cosmetic field

[0100] 1. The preparation method of the human iPSC-derived platelet-rich suspension is as described in Examples 1 to 4.

[0101] 2. The above suspension is used in the cosmetic field.

[0102] 3. The treatment methods are: direct injection (dermal 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.

[0103] 4. Precautions for treatment:

[0104] The treatment with the 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:

[0105] (1) Women should prepare and use concentrated platelet products during non-menstrual periods. Pregnant, lactating, and women planning pregnancy, as well as patients with anemia, abnormal blood coagulation function, and abnormal liver function are prohibited;

[0106] (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; sweating and massage are not recommended 1 week after treatment.

[0107] Example 9: Application of platelet suspension in the treatment of hair loss

[0108] 1. The preparation method of the human iPSC-derived platelet-rich suspension is as described in Examples 1 to 4.

[0109] 2. The above suspension is used for the treatment of hair loss.

[0110] 3. The treatment methods are as follows: direct multi-point injection (dermis, superficial subcutaneous layer), application, and combined use at the alopecia positions. Depending on different application directions, it can be referred to that the treatment is carried out once every 1 - 2 months, and 3 - 4 times for each course of treatment.

[0111] 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.

[0112] 5. Treatment conclusion:

[0113] 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, proving 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.

[0114] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. All the documents mentioned in the present invention are incorporated herein by reference in their entirety. 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 alopecia, 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 composition is directly administered by multi-point injection, smearing, and / or combined use of injection and smearing at the alopecia positions of the dermis layer and the superficial subcutaneous layer.

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

3. A composition for treating alopecia, 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; wherein, the aforementioned composition is applied to the alopecia positions in the dermal layer and the superficial subcutaneous layer by directly performing multi-point injection, smearing, and / or combined use of injection and smearing.

4. The composition for treating alopecia 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 alopecia according to claims 1 to 3, characterized in that, the composition is administered once every 1 to 2 months, and every 3 to 4 times is a treatment course.

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

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