Suspension and gel for preparing platelet-rich cells through in-vitro differentiation of human pluripotent induced stem cells (iPSC) and preparation method of suspension and gel

Abstract

The invention provides a platelet preparation prepared by differentiation of human pluripotent induced stem cells (iPSC), and a preparation method of the platelet preparation. The platelet preparation is prepared by differentiation of the human pluripotent induced stem cells (iPSC). Compared with a platelet-rich plasma (PRP) preparation with blood separation, the platelet preparation provided by the invention has the advantages that the ingredients are relatively stable, and the slow-release growth factors are beneficial to tissue repair. In addition, the preparation method disclosed by the invention can be used for large-scale preparation and standardized production, and the functional platelet-rich suspension / gel with stable batches can be produced.

Description

Technical Field

[0001] The present invention relates to the field of biomedical materials, and relates to a method for preparing platelets in vitro, and particularly relates to a platelet-rich suspension and / or gel prepared based on the in vitro differentiation of human induced pluripotent stem cells (iPSCs) and a preparation method thereof. Background Art

[0002] The basic physiological function of platelets is to coagulate and stop bleeding by adhering to the damaged site and forming a thrombus during vascular injury. Clinically, in addition to conventional transfusable apheresis platelets or concentrated platelets, platelet concentrates such as platelet-rich plasma (PRP) and platelet-rich fibrin (PRF) have also received extensive attention. For the direction of PRP and PRF, the role of platelet-derived growth factors is the focus of application. Because the alpha granules of platelets can release a large number of growth factors and cytokines, such as: platelet-derived growth factor (PDGF), such as PDGFαα, PDGFββ and PDGFαβ; transforming growth factor beta (TGFβ), such as TGFβ1 and TGFβ2; vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF), etc.

[0003] After platelet activation, the release of growth factors can increase several-fold in a short time and produce corresponding biological functions, such as: promoting the regeneration of epithelium, blood vessels and bone, promoting the synthesis of collagen, promoting cell proliferation, and affecting the migration and differentiation of stem cells, etc.; in addition, platelet activation also causes changes in the content of some cytokines related to immune regulation and immune response, such as IL-1beta, IL-8, etc., and affects the expression of some proteins related to blood homeostasis, such as activated sequence proteins and adhesion proteins, etc.

[0004] PRP and PRF are 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, liquefaction of skin and adipose tissue, etc.); and dentistry (periodontal disease, jaw surgery, etc.). However, since they are generally prepared from autologous blood sampling, they are often restricted and affected by 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 hospital systems and doctors' experience. In addition, since the preparation processes of PRP and PRF are both carried out in vitro, there will also be some operation-related risks such as infection.

[0005] The preparation of platelets differentiated from induced pluripotent stem cells (iPSC) is a prerequisite for producing a homogenized platelet concentrate product and also a prerequisite for establishing corresponding treatment standards. The preparation of iPSC platelets is achieved through the in vitro differentiation of iPSC, and the entire in vitro differentiation and regulation pathway is 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.

[0006] However, existing technologies still lack methods for large-scale preparation, stable quality for each batch, and standardized production in the preparation of functional platelet-rich plasma and its preparations. Summary of the Invention

[0007] In this invention, platelets are differentiated from human induced pluripotent stem cells (iPSC) in vitro and produced using a reactor. Then, the platelets are activated in vitro to increase the release of their growth factors and cytokines, ultimately providing a method for preparing a functional platelet-rich suspension / gel that can be produced in large quantities, has stable batches, and can be produced standardized.

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

[0009] The present invention provides a platelet preparation, comprising the following components:

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

[0011] (2) Additives: Normal saline of 0.9% NaCl and 5% human recombinant serum albumin.

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

[0013] The present invention also provides a platelet preparation, 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.

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

[0017] Preferably, the pluripotent induced stem cells of the present invention are human pluripotent induced stem cells.

[0018] Preferably, the above-mentioned platelet preparation can be applied to the treatment of arthritis, tennis elbow, promoting wound healing, treating alopecia, etc., and applied to the beauty field.

[0019] In the examples, the present invention also provides a method for preparing a platelet preparation, comprising the following steps:

[0020] (1) Culturing and differentiating pluripotent induced stem cells into hematopoietic precursor stem cells through the first culture condition, the second culture condition, and the third culture condition.

[0021] (2) Culturing and differentiating the above-mentioned hematopoietic precursor stem cells into megakaryocytes, transferring the megakaryocytes to a reactor to produce platelets;

[0022] (3) Concentrating the platelets;

[0023] (4) Adding additives.

[0024] Preferably, the above-mentioned additives include normal saline of 0.9% NaCl and 5% human recombinant serum albumin.

[0025] More preferably, the above-mentioned additives are freeze-dried thrombin of 100 U / mL, 1% calcium gluconate, and 0.05% type I collagenase.

[0026] Preferably, the above-mentioned additives include 10 μM ADP, 10 - 100 μg / mL fibrinogen, 2 mM CaCl 2 and 0.05 - 0.5% type I collagenase.

[0027] Preferably, the first culture condition is:

[0028] (1) Culture for 2 days;

[0029] (2) Add 30 ng / mL VEGF-A165, 10 ng / mL bFGF, 5 μM Y-27632, 2 mM Chir-99021, 20 ng / mL Activin A to Essential 8 medium.

[0030] Preferably, the second culture condition is:

[0031] (1) Culture for 4 days;

[0032] (2) Replace the medium with HDM medium and add 30 ng / mL VEGF-

[0033] A165, 10 ng / mL bFGF, 20 ng / mL BMP4, 5 μM Y-27632,

[0034] 20 ng / mL Activin A.

[0035] Preferably, the third culture condition is:

[0036] (1) Culture for 8 days;

[0037] (2) Replace the medium with HDM medium and add 30 ng / mL VEGF-

[0038] A165, 10 ng / mL bFGF, 50 ng / mL SCF, 50 ng / ML thrombopoietin, 2 U / mL EPO, 5 μM Y-27632.

[0039] Preferably, to culture and differentiate the above hematopoietic precursor stem cells into megakaryocytes, the following need to be added:

[0040] (1) Culture for 15 - 20 days;

[0041] (2) Add 20 ng / mL VEGF-A165, 5 ng / mL bFGF, 50 ng / mL thrombopoietin, 20 ng / mL IL-3, 20 ng / mL IL-6, 50 ng / mL IL-21,

[0042] 5 nM Tazemetostat, 200 nM Eltrombopag, 200 nM iBET151.

[0043] More preferably, the culture condition for the proliferation of the above megakaryocytes is:

[0044] (1) The seeding density is 5×106 / mL;

[0045] (2) Replace the culture medium with HDM supplemented with 50 ng / mL thrombopoietin,

[0046] 50 ng / mL stem cell factor, 50 ng / mL IL-21, 5 μM Y-27632.

[0047] Preferably, before using the above reactor, add 50 ng / mL thrombopoietin, 0.5 ng / mL collagen, 5 nM Fingolimod HCl, 5 μM Y-276325 to promote platelet maturation.

[0048] More preferably, after thawing and resuscitating the above megakaryocytes and before using the above reactor, 0.5 ng / mL collagen and 5 nM Fingolimod HCl can be added.

[0049] Preferably, the above reactor includes a multi-layer movable grid structure with a variable rotational motion mode.

[0050] More preferably, the rotational motion mode of the above reactor is selected from the following two alternatives:

[0051] (1) The grid is static and the liquid is dynamic;

[0052] (2) The grid is dynamic and the liquid flow is static.

[0053] More preferably, the rotational speed of the above reactor is 120 - 150 rpm, and the maximum linear velocity of the liquid flow is 30 cm / second.

[0054] The present invention also provides a pharmaceutical composition containing an effective amount of the above platelet preparation and a pharmaceutically acceptable carrier.

[0055] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0056] (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 CD34 + cell purity.

[0057] (2) During the process of differentiating hematopoietic precursor 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.

[0058] (3) After cryopreservation and resuscitation of megakaryocytes and before using the reactor, adding 0.5 ng / mL collagen and 5 nM Fingolimod HCl helps promote platelet maturation.

[0059] (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

[0060] release platelets.

[0061] (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.

[0062]

[0063] (6) The preparation methods (suspension formula, activated suspension preparation formula, gel preparation

[0064] formula and preparation method) have not been used in the application scenarios of iPSC-platelets.

[0065] BRIEF DESCRIPTION OF THE DRAWINGS

[0066] 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:

[0067] Figure 1 are the morphologies (a), cell sizes (b) of hematopoietic progenitor cells (HPC) generated from induced pluripotent stem cells (iPSC), and CD34 + cells (c) and their purities (d) obtained by passage purification. CD34: A marker mainly expressed on hematopoietic stem cells and hematopoietic progenitor cells. BF: bright field, bright field view.

[0068] Figure 2 are the data comparisons of iPSC-derived (a) 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).

[0069] 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 can occur within 72 hours in the reactor (d). Through flow cytometry detection, it is found that iPSC-derived platelets (iPLTs) have a similar marker expression (f) compared to peripheral blood-derived platelets (e). BF: bright field, bright field view. PB-PLTS: Peripheral blood-derived platelets.

[0070] Figure 4 For the states of unactivated and activated platelet suspensions during the preparation process of platelet suspensions. Detailed implementation manners

[0071] 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 specific implementation manners, rather than 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 thought of 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.

[0072] 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, the meanings of 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.

[0073] 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 rest of the content disclosed by the present invention in any way.

[0074] In order to provide a platelet preparation with stable components, as well as a preparation method that can be prepared in large quantities, has stable batches, and can be produced standardly, the present invention utilizes the iPSC platelet preparation method, reactor, suspension formula, activated suspension formula, gel formula and preparation method to achieve technical effects.

[0075] The present invention provides a method for preparing platelets using stem cells. First, the process of differentiating pluripotent induced stem cells (iPSC) into hematopoietic precursor stem cells (HPC) is divided into three stages, and each stage has its own corresponding culture medium formula:

[0076] Table 1.

[0077]

[0078] 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.

[0079] 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.

[0080] The present invention uses a reactor to produce 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.

[0081] 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 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-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 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.

[0082] The formula of the suspension and gel additives of the present invention is as follows:

[0083] Table 2.

[0084]

[0085]

[0086] In addition, the iPSC platelet preparation of the present invention can be applied to different scenarios, such as treating arthritis, tennis elbow, promoting wound healing, treating hair loss, etc.; it can also be applied to the beauty field.

[0087] Example 1: Differentiating iPSCs into hematopoietic progenitor cells (iHPCs)

[0088] The iPSCs of the present invention were obtained from skin biopsies in the hospital, and the primary fibroblasts were cultured in the laboratory and screened after reprogramming to obtain an ideal iPSC cell line.

[0089] 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.

[0090] 2. Then change the culture medium to HDM, and the formula is as follows:

[0091] 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).

[0092] 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.

[0093] 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.

[0094] 4. Collect hematopoietic progenitor cells (iHPCs) growing in suspension, collect the culture medium supernatant containing iHPCs, centrifuge at 1000 rpm for 5 minutes.

[0095] The results are shown in Figure 1 . The hematopoietic progenitor cells (HPCs) generated from iPSCs in this example Figure 1 a) have a cell size similar to that of CD34 + cells separated from peripheral blood ( Figure 1 b), and through subculture and 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..

[0096] Example 2: Differentiation and proliferation of megakaryocytes

[0097] I. Differentiate hematopoietic progenitor cells (iHPC) into megakaryocytes (iMKs)

[0098] 1. Collect the hematopoietic progenitor cells obtained in Example 1, seed them in a culture dish pre-coated with 0.1% gelatin, and 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 to culture for 15 - 20 days.

[0099] 2. Collect the suspended megakaryocytes.

[0100] II. In vitro proliferation of megakaryocytes (iMKs)

[0101] 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.

[0102] 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.

[0103] 3. Megakaryocytes can be cryopreserved. The cryopreservation solution is HDM + 5% BSA + 5% DMSO. After programmed cryopreservation, they are stored in liquid nitrogen.

[0104] The results are shown in Figure 2 . Two different background iPSCs ( Figure 2 a) megakaryocytes (iMKs) 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).

[0105] Example 3: Production of platelets

[0106] 1. Suspend the megakaryocytes obtained in Example 2 in PM medium, and add 50 ng / mL thrombopoietin (TPO) and 5 μM Y-27632, and statically culture for 1 day.

[0107] PM medium: Add 1x ITS, 1x glutamax, 0.45 mM monothioglycerol, 50 μg / mL ascorbic acid, 10 U heparin, 5% human plasma to Iscove modified Dulbecco (IMDM).

[0108] 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.

[0109] 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.

[0110] 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 rotatable movement mode in variable directions, a rotation speed of 120 - 150 rpm, and the maximum linear velocity of the liquid flow is 30 cm / s.

[0111] 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 was found that intense megakaryocyte fragmentation and platelet release can occur in the reactor within 72 hours ( Figure 3 d). Through flow cytometry detection, it was found that platelets derived from iPSCs (iPLTs) and platelets from peripheral blood ( Figure 3 e) have similar marker expression ( Figure 3 f) conditions.

[0112] Example 4: Preparation methods of platelet suspension and gel

[0113] 1. Platelet-rich suspension derived from human iPSCs

[0114] 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.

[0115] 2. Activated platelet-rich suspension derived from human iPSCs

[0116] 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.

[0117] 3. Platelet-rich gel derived from human iPSCs

[0118] 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 to obtain the lower layer rich in activated platelet gel.

[0119] The results are shown as follows Figure 4 During the preparation of the platelet suspension, the unactivated platelet suspension (described in Method 1 of this Example) presented 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.

[0120] Example 5: Application of platelet suspension in the treatment of arthritis

[0121] 1. The preparation method of the platelet-rich suspension derived from human iPSCs was as described in Examples 1-4.

[0122] 2. The above-mentioned platelet suspension was used to treat arthritis, specifically including osteoarthritis and rheumatoid arthritis.

[0123] 3. The injection method was as follows: intra-articular injection into the knee joint under ultrasound guidance, using a 22-gauge needle, 5 mL of fresh platelet-rich suspension derived from human iPSCs each time. After injection, the knee joint was flexed and extended passively 5 times, and the patient rested for 10 minutes. Injection was performed once a week, 3 times as a course, and the patients were followed up within 12 months after injection.

[0124] 4. The evaluation method for treatment improvement was as follows: 1) average knee joint pain score (11-point numerical rating scale, 0 = no pain - 10 = most severe pain); 2) measurement of the medial tibial cartilage volume by MRI; 3) 31 auxiliary evaluation indicators (25 related to symptoms, 6 for MRI evaluation) to evaluate pain, function, quality of life, overall changes, and joint structure, etc.

[0125] Example 6: Application of platelet suspension in the treatment of tennis elbow

[0126] 1. The preparation method of the platelet-rich suspension derived from human iPSCs was as described in Examples 1-4.

[0127] 2. The above-mentioned platelet suspension was used to treat tennis elbow.

[0128] 3. The treatment method was as follows: The injection site was blocked with 0.5% bupivacaine and adrenaline, and then 2-3 mL of the prepared platelet-rich suspension derived from human iPSCs was injected into the extensor carpi radialis brevis tendon and the surrounding area by the tendon-pricking method. The needle was inserted once without being withdrawn and the direction was changed 5 times to inject the tendon for infiltration.

[0129] 4. The evaluation method for treatment improvement is as follows: 1) At 4, 8, 12, 16, 20, and 24 weeks after treatment, a pain score improvement of 25% or more in the VAS pain score for wrist extension (evaluating the severity of pain using the visual analogue method, VASRWE) compared to the baseline is considered a success; 2) Auxiliary evaluation indicators: the patient-rated tennis elbow evaluation (PRTEE); extended wrist examination.

[0130] Example 7: Application of platelet suspension in wound healing treatment

[0131] 1. The preparation method of human iPSC-derived platelet-rich suspension is as in Examples 1 to 4.

[0132] 2. The above suspension is used to promote wound healing, such as in the healing of trauma and burns.

[0133] 3. The treatment method is: after surgical treatment of trauma, before closing and suturing the incision, the iPSC-derived platelet-rich suspension is applied 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 trauma area and degree of injury.

[0134] 4. The evaluation indicators for treatment improvement are: 1) Wound healing time; 2) Wound infection assessment; 3) Wound healing quality.

[0135] For general wounds, unactivated platelet PRP is used to slowly release growth factors to promote healing, while for severely damaged or infection-prone traumas, activated platelets are used so that a large amount of growth factors are enriched and rapidly released at the trauma site in a short time, thereby rapidly promoting injury healing.

[0136] Example 8: Application of platelet suspension in the cosmetic field

[0137] 1. The preparation method of human iPSC-derived platelet-rich suspension is as in Examples 1 to 4.

[0138] 2. The above suspension is used in the cosmetic field.

[0139] 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 treatment is carried out once every 1 to 2 months, and 3 to 4 times for each course of treatment.

[0140] 4. Precautions for treatment:

[0141] The treatment with human iPSC-derived platelet-rich suspension does not require a recovery period, and patients can go to work as normal on 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:

[0142] (1) Women should prepare and use concentrated platelet products during non-menstrual periods. It is contraindicated for pregnant, lactating, and women planning pregnancy, as well as patients with anemia, abnormal coagulation function, and abnormal liver function;

[0143] (2) Keep the treatment area dry for 24 hours after treatment, do not get it wet, and 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.

[0144] Example 9: Application of platelet suspension in the treatment of hair loss

[0145] 1. The preparation method of human iPSC-derived platelet-rich suspension is as described in Examples 1 to 4.

[0146] 2. Use the above suspension for the treatment of hair loss.

[0147] 3. The treatment methods are: direct multi-point injection (dermis, superficial subcutaneous layer), application, and combined use at the hair loss sites. 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.

[0148] 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.

[0149] 5. Treatment conclusion:

[0150] 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 result of the 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.

[0151] 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 principles of the present invention, and these equivalent modifications also fall within the scope defined by the claims of this application.

Claims

1. A platelet preparation, characterized in that, it comprises the following components: (1) Platelet concentrate prepared by differentiating pluripotent induced stem cells; (2) Additives: physiological saline of 0.9% NaCl and 5% human recombinant serum albumin.

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

3. A platelet preparation, 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 platelet preparation according to any one of claims 1 to 3, characterized in that, The concentration of the platelet concentrate is 5 to 20×10 11 / L.

5. A method for preparing a platelet preparation, characterized in that, it comprises the following steps: (1) Culturing and differentiating pluripotent induced stem cells into hematopoietic precursor stem cells through a first culture condition, a second culture condition, and a third culture condition; (2) Culturing and differentiating the hematopoietic precursor stem cells into megakaryocytes, transferring the megakaryocytes to a reactor to produce platelets; (3) Concentrating the platelets to obtain a platelet concentrate differentiated from pluripotent induced stem cells; (4) Adding additives.

6. The method according to claim 5, characterized in that, the first culture condition is Essential 8 medium supplemented with 30 ng / mL VEGF-A165, 10 ng / mL bFGF, 5 μM Y-27632, 2 mM Chir-99021, 20 ng / mL Activin A.

7. The method according to claim 5, characterized in that, the second culture condition is HDM medium supplemented with 30 ng / mL VEGF-A165, 10 ng / mL bFGF, 20 ng / mL BMP4, 5 μM Y-27632, 20 ng / mL Activin A.

8. The method according to claim 5, characterized in that, the third culture condition is HDM medium supplemented with 30 ng / mL VEGF-A165, 10 ng / mL bFGF, 50 ng / mL SCF, 50 ng / ML thrombopoietin, 2 U / mL EPO, 5 μM Y-27632.

9. The method according to claim 5, characterized in that, when the hematopoietic precursor stem cells are cultured and differentiated into megakaryocytes, 50 ng / mL IL-21, 5 nM Tazemetostat, 200 nM Eltrombopag, and 200 nM iBET151 need to be added.

10. The method according to claim 5, characterized in that, the additives comprise physiological saline of 0.9% NaCl and 5% human recombinant serum albumin.

11. The method according to claim 10, characterized in that, the additives further comprise freeze-dried thrombin of 100 U / mL, 1% calcium gluconate and 0.05% type I collagenase.

12. The method according to claim 5, characterized in that, The additive contains 10 μM ADP, 10-100 μg / mL fibrinogen, 2 mM CaCl 2 and 0.05-0.5% type I collagenase.

13. The method according to claim 5, characterized in that, the reactor comprises a multi-layer movable grid structure, and its rotational movement mode is variable.

14. A pharmaceutical composition, characterized in that, it contains an effective amount of the platelet preparation according to claim 1 or 3 and a pharmaceutically acceptable carrier.

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