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

Abstract

The invention provides a composition for promoting wound healing, 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 promoting wound healing. 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 to the application of an iPSC-derived platelet-rich material prepared in vitro in wound healing treatment. Background Art

[0002] Tissue damage caused by trauma triggers acute inflammation. Similar to inflammatory cells, platelets play different roles in the processes of hemostasis, antibacterial, and tissue repair after trauma. The basic function of platelets is to stop bleeding caused by trauma, and the Toll-like receptors on the platelet surface can also sense pathogens and release thrombocidins 1-2 and thymosin β4 in the α granules to play a bactericidal role, cooperating with neutrophils and macrophages to remove damaging factors. After the damaging factors are removed, the inflammation enters the process of tissue repair and regeneration. Granulation tissue is a precursor medium for tissue repair, which contains newly formed capillary tissue and loose connective tissue. As the repair process progresses, the granulation tissue is gradually transformed into mature connective tissue to complete tissue repair. Platelets release VEGF, FGF, PDGF, angiostatin, endostatin, and S1P during this period to induce angiogenesis and stabilize new blood vessels, providing nutrients and raw materials for tissue repair. At the same time, platelets also secrete EGF, TGF-β, HGF, and insulin-like growth factor 1 (IGF-1) to promote tissue repair.

[0003] Traditional peripheral blood platelet-rich plasma (PRP) has been clinically applied in the process of promoting wound healing. Platelets in PRP release different growth factors like a sustained-release drug to promote wound healing. However, currently, PRP is prepared from donated peripheral blood, with a single and limited source. The platelet-rich suspension derived from iPSC can provide stable and large amounts of platelets to meet clinical needs. In addition, through gene editing and post-induction treatment, not only can the overall expression level of platelet growth factors be increased, but also the types and contents of growth factor expression can be adjusted, making the treatment more controllable and effective at different times of wound healing. For example, a platelet-rich suspension expressing TGF-β1 and TGF-β2 is given during the acute inflammation period; a platelet-rich suspension expressing PDGF, FGF, and VEGF is given during the wound healing period; and a platelet-rich suspension expressing TGF-β3 is given during the tissue remodeling period in the late stage of healing.

[0004] The preparation of iPSC - platelets is a prerequisite for producing a homogenized platelet concentrate product and also for establishing corresponding treatment standards. The preparation of iPSC platelets is achieved through the in vitro differentiation of iPSCs, and the in vitro differentiation and regulation pathways throughout the process are currently relatively clear. In 2018, the team of Koji Eto at Kyoto University in Japan reported 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.

[0005] However, in the aspect of wound healing treatment, the existing technology still lacks functional platelet - rich preparations that can be produced in large quantities, have stable quality for each batch, and can be produced in a standardized manner. Summary of the Invention

[0006] The present invention provides a method for in vitro differentiating human induced pluripotent stem cells (iPSCs) and using a reactor to produce platelets and activate them in vitro, and finally provides a platelet composition for promoting wound healing.

[0007] To achieve the above - mentioned purpose, the present invention provides the following technical solutions:

[0008] The present invention provides a composition for promoting wound healing, comprising the following components:

[0009] (1) A platelet concentrate prepared by differentiating pluripotent induced stem cells;

[0010] (2) Additives: physiological saline of 0.9% NaCl and 5% human recombinant serum albumin.

[0011] Among them, the application dose of the aforementioned composition is 5 mL each time.

[0012] The present invention also provides a composition for promoting wound healing, comprising the following components:

[0013] (1) A platelet concentrate prepared by differentiating pluripotent induced stem cells;

[0014] (2) Additives: 10 μM ADP, 10 - 100 μg / mL fibrinogen, 2 mM CaCl2, and 0.05 - 0.5% type I collagenase; among them, the application dose of the composition is 5 mL each time. Preferably, the concentration of the above - mentioned platelet concentrate is 5 - 20×10 11 / L.

[0015] Preferably, the platelet content of the above - mentioned composition is not less than 1×10 6 / μL.

[0016] More preferably, the above composition is used in the preparation of a drug for promoting wound healing.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] (1) The process of differentiating iPSCs into hematopoietic progenitor stem cells is divided into three stages, each stage having its own corresponding culture medium formula, which helps to improve the purity of CD34 + cells.

[0019] (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.

[0020] (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.

[0021] (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 produce intense megakaryocyte fragmentation and help release platelets.

[0022] (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.

[0023] (6) The preparation methods (suspension formula, activated suspension preparation formula, gel preparation formula and preparation method) have never been used in the application scenario of iPSC-platelets.

[0024] (7) It can efficiently produce platelet-derived growth factor, and its properties are stable in batches, which can produce effective therapeutic effects and generate clinical data that can promote wound healing. Description of the Drawings

[0025] 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 constitute an improper limitation of the present invention. In the drawings:

[0026] Figure 1 are the morphology (a) of hematopoietic progenitor stem cells (Hematopoietic progenitor cells) generated from pluripotent induced stem cells (iPSCs), the cell size (b) of hematopoietic stem cells, and CD34 obtained by 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.

[0027] Figure 2 Data comparison of iPSC-derived megakaryocytes (iMKs) from a newborn male (A) and a 34-year-old female (B) after cryopreservation and recovery, including morphology (b, c), proliferation ability (d, e), cell viability (f), and cell size (g).

[0028] Figure 3 For 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, intense megakaryocyte fragmentation and platelet release were found to occur within 72 hours in the reactor (d). Through flow cytometry, it was found that iPSC-derived platelets (iPLTs) had similar marker expression (f) compared to peripheral blood-derived platelets (e). BF: bright field, bright field view. PB-PLTS: Peripheral blood-derived platelets.

[0029] Figure 4 For the state of platelet suspensions before and after activation during the preparation process of platelet suspensions. Detailed implementation manners

[0030] The following further describes the present invention in combination 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 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.

[0031] All technical and scientific terms used herein have the same meaning 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 embodiments are common knowledge and well-known to those skilled in the art. The embodiments in the present application and the features in the embodiments can be combined with each other.

[0032] The characteristics and advantages of the present invention can be further understood through the following detailed description in combination 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.

[0033] 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 achieves technical effects by using the iPSC platelet preparation method, reactor, suspension formulation, activated suspension formulation, gel formulation, and preparation method.

[0034] 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:

[0035] Table 1.

[0036]

[0037] 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.

[0038] 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 of Example 3), and add 0.5 ng / mL Collagen and 5 nM Fingolimod HCl to promote platelet maturation.

[0039] 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.

[0040] Although the method for preparing iPSC platelet preparations in the present invention refers to the traditional PRP preparation concept, 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, there are significant differences in their components and proportions, 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 very good guiding and reference role in 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-derived 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.

[0041] The suspension and gel additives of the present invention are formulated as follows:

[0042] Table 2.

[0043]

[0044] 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 alopecia, etc.; they can also be applied to the beauty field.

[0045] Example 1: Differentiate iPSC into hematopoietic progenitor cells (iHPCs)

[0046] 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.

[0047] 1. Dissociate iPSC 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.

[0048] 2. Then change the culture medium to HDM, with the following formula:

[0049] 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).

[0050] 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 culturing for 4 days.

[0051] 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 culturing for 8 days.

[0052] 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.

[0053] For the results, please refer to Figure 1 . The hematopoietic precursor stem cells (Hematopoietic progenitor 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 + cells ( Figure 1 b), and through passage purification, the obtained CD34 + cells ( Figure 1 c) can reach a purity of about 90% ( Figure 1 d). More starting materials help reduce interference in the subsequent differentiation process and do not generate excessive heterologous cells.

[0054] Example 2: Differentiation and proliferation of megakaryocytes

[0055] I. Differentiate hematopoietic precursor stem cells (iHPC) into megakaryocytes (iMKs)

[0056] 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.

[0057] 2. Collect the suspended megakaryocytes.

[0058] II. In vitro proliferation of megakaryocytes (iMKs)

[0059] 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.

[0060] 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.

[0061] 3. Megakaryocytes can be cryopreserved. The cryopreservation solution is HDM + 5% BSA + 5% DMSO, and after programmed freezing, it is stored in liquid nitrogen.

[0062] The results are shown in Figure 2 . Megakaryocytes (iMKs) from two different background iPSCs Figure 2 a) derived 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 2 c), a proliferation ability of 10,000-fold in 15 days ( Figure 2 d, Figure 2 e), as well as relatively stable cell viability ( Figure 2 f) and cell size ( Figure 2 g).

[0063] Example 3: Production of platelets

[0064] 1. Suspend the megakaryocytes obtained in Example 2 in PM medium, and add 50 ng / mL thrombopoietin (TPO) and 5 μM Y-27632, and incubate statically for 1 day.

[0065] 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).

[0066] 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, incubate statically 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.

[0067] 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 in an environment of 37°C and 5% CO 2 , and the maximum horizontal liquid streamline velocity in the reactor is 30 cm / s.

[0068] 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.

[0069] For the results, see 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 3 d). Through flow cytometry detection, it is found that iPSC-derived platelets (iPLTs) and platelets from peripheral blood( Figure 3 e) have similar marker expression( Figure 3 f) conditions.

[0070] Example 4: Preparation method of platelet suspension and gel

[0071] 1. Platelet-rich suspension derived from human iPSCs

[0072] 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 thereto.

[0073] 2. Activated human iPSC-derived platelet-rich suspension

[0074] 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.

[0075] 3. Human iPSC-derived platelet-rich gel

[0076] 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 stand and crosslink for 5 minutes. Centrifuged at 200 rpm for 15 minutes, and then continued to centrifuge at 1500 rpm for 2 minutes. The supernatant was discarded, and the lower layer rich in activated platelet gel could be obtained.

[0077] For the results, please see 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.

[0078] Example 5: Application of platelet suspension in the treatment of arthritis

[0079] 1. The preparation method of the human iPSC-derived platelet-rich suspension was as in Examples 1 - 4.

[0080] 2. The above platelet suspension was used to treat arthritis, specifically including osteoarthritis and rheumatoid arthritis.

[0081] 3. The injection method was: 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 was passively flexed and extended 5 times, and the patient rested for 10 minutes. Injected once a week, 3 times per course of treatment, and the patients were followed up within 12 months after injection.

[0082] 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) 31 auxiliary evaluation indicators (25 related to symptoms and 6 for MRI evaluation) to evaluate pain, function, quality of life, overall changes, and joint structure, etc.

[0083] Example 6: Application of platelet suspension in the treatment of tennis elbow

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

[0085] 2. The above platelet suspension is used to treat tennis elbow.

[0086] 3. The treatment method is as follows: The injection site is blocked with 0.5% bupivacaine and adrenaline, and then 2 - 3 mL of the prepared human iPSC-derived platelet-rich suspension is injected into the extensor carpi radialis brevis tendon and the surrounding area by the tendon puncture method. The needle is inserted once without being withdrawn and the direction is changed 5 times to inject the tendon for infiltration.

[0087] 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 (judging 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.

[0088] Example 7: Application of platelet suspension in the treatment of wound healing

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

[0090] 2. The above suspension is used to promote wound healing, such as for trauma and burn healing.

[0091] 3. The treatment method is as follows: After surgical treatment of the trauma, before closing and suturing the incision, the iPSC-derived platelet-rich suspension is applied into the wound, 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.

[0092] 4. The evaluation indicators for treatment improvement are as follows: 1) Wound healing time; 2) Wound infection assessment; 3) Wound healing quality.

[0093] For general wounds, unactivated platelet PRP is used to enable the slow release of growth factors to promote healing. For severe or infection-prone injuries, 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.

[0094] Example 8: Application of platelet suspension in the field of beauty

[0095] 1. The preparation method of human iPSC-derived platelet-rich suspension is as described in Examples 1 to 4.

[0096] 2. The above suspension is used in the field of beauty.

[0097] 3. The treatment methods are: direct injection (dermis, 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.

[0098] 4. Precautions for treatment:

[0099] 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. After treatment, swelling or bleeding points may appear in the treatment area. 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:

[0100] (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 coagulation function, and abnormal liver function, are prohibited from using;

[0101] (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; do not recommend sweating and massage 1 week after treatment.

[0102] Example 9: Application of platelet suspension in the treatment of hair loss

[0103] 1. The preparation method of human iPSC-derived platelet-rich suspension is as described in Examples 1 to 4.

[0104] 2. The above suspension is used for the treatment of hair loss.

[0105] 3. The treatment methods are: direct multi-point injection (dermis, 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.

[0106] 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.

[0107] 5. Treatment conclusion:

[0108] 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, demonstrating a clear improvement in hair strength; in addition, histopathological evaluation showed that after injection of iPSC-derived platelet-rich suspension, the epidermal thickness and the number of hair follicles in the scalp increased significantly.

[0109] 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 principles of the present invention, and these equivalent forms of modifications also fall within the scope defined by the claims of this application.

Claims

1. A composition for promoting wound healing, 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 dosage of the composition is 5 mL each time.

2. The composition for promoting wound healing 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 promoting wound healing, 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 administration dose of the composition is 5 mL each time.

4. The composition for promoting wound healing 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 promoting wound healing according to claims 1 to 3, characterized in that, The platelet content of the composition is not less than 1×10 6 / μL.

6. Use of the composition according to any one of claims 1 to 3 in the preparation of a drug for promoting wound healing.

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