Cell-based formulation containing pluripotent stem cells for diseases or post-acute sequelae caused by SARS-cov-2 infection

Abstract

This cell-based formulation contains SSEA-3-positive pluripotent stem cells derived from mesenchymal tissue of a living body or derived from cultured mesenchymal cells. This cell-based formulation is characterized in that the formulation is for administration to address diseases and / or post-acute sequelae caused by SARS-CoV-2 infection. The present invention makes it possible to provide a cell-based formulation that contains pluripotent stem cells and is used for treating and / or preventing SARS-CoV-2 infection-caused diseases such as pneumonia and pulmonary fibrosis and SARS-CoV-2 infection-caused post-acute sequelae such as olfactory dysfunctions.

Description

Cell therapies containing pluripotent stem cells for diseases or post-symptomatic symptoms caused by SARS-CoV-2 infection.

[0001] The present invention relates to a cell preparation containing pluripotent stem cells for diseases or post-symptomatic symptoms caused by SARS-CoV-2 infection.

[0002] In recent years, in the field of regenerative medicine, cell therapy using stem cells has been studied for various diseases, and embryonic stem cells (ES cells), neural stem / progenitor cells (NSPCs), induced pluripotent stem cells (iPS cells), and umbilical cord blood stem cells (UCBCs) are known as stem cells that have the potential for clinical application.

[0003] Furthermore, bone marrow mesenchymal cell fractions (MSCs), isolated from adults, are known to have the ability to differentiate into, for example, bone, cartilage, adipocytes, nerve cells, and skeletal muscle (Non-Patent Literature 1 and 2). However, MSCs are a group of cells that include various types of cells, and the nature of their differentiation potential is not well understood, resulting in considerable variability in therapeutic effects. In addition, while iPS cells have been reported as pluripotent stem cells derived from adults, establishing iPS cells requires extremely complex procedures, such as introducing specific genes or compounds into somatic cells in the dermal fibroblast fraction, which is a mesenchymal cell. Moreover, iPS cells have a high tumor-forming capacity, creating extremely high hurdles to clinical application.

[0004] Research by Izawa, one of the inventors, has revealed that pluripotent stem cells (Multilineage-differentiating Stress Enduring cells; Muse cells) that are present in the mesenchymal cell fraction and can be obtained without induction, expressing SSEA-3 (Stage-Specific Embryonic Antigen-3) as a surface antigen, are responsible for the pluripotency of the mesenchymal cell fraction and may have potential applications in disease treatment aimed at tissue regeneration (Patent Document 1).

[0005] Japan has the world's highest life expectancy, and it is known that when the very elderly are infected with SARS-CoV-2, the mortality rate and severity rate are overwhelmingly higher than in younger people. Therefore, countermeasures against SARS-CoV-2 infection remain a priority for healthcare workers who have frequent contact with the very elderly. Furthermore, there have been reports of various post-symptomatic symptoms developing after SARS-CoV-2 infection. While many of these symptoms tend to improve over time, some have resulted in significant limitations on social life. As mentioned above, Muse cells are considered to have potential applications in disease treatment aimed at tissue regeneration, but there have been no examples of their application to diseases caused by SARS-CoV-2 infection or to post-symptomatic symptoms.

[0006] Patent No. 5185443

[0007] Dezawa, M. , et al. , J. Clin. Invest. , Vol. 113, p. 1701-1710, 2004 Dezawa, M. , et al. , Science, Vol. 309, p. 314-317, 2005

[0008] The present invention was made to solve the above problems and aims to provide a cell preparation containing pluripotent stem cells that can be used to treat and / or prevent diseases such as pneumonia and pulmonary fibrosis caused by SARS-CoV-2 infection, as well as post-symptomatic symptoms such as olfactory dysfunction.

[0009] The inventors of this invention have diligently studied and completed the present invention in order to solve the above problems. That is, the present invention is as follows: [1] to [7].

[0010] [1] A cell preparation comprising SSEA-3 positive pluripotent stem cells derived from living mesenchymal tissue or cultured mesenchymal cells, characterized in that it is used to be administered for diseases and / or post-symptomatic symptoms caused by SARS-CoV-2 infection; [2] The cell preparation according to [1], wherein the pluripotent stem cells are used to treat and / or prevent diseases and / or post-symptomatic symptoms caused by SARS-CoV-2 infection; [3] The cell preparation according to [1] or [2], wherein the disease is pneumonia and / or pulmonary fibrosis; [4] The cell preparation according to any one of [1] to [3], wherein the post-symptomatic symptoms are olfactory dysfunction; [5] The pluripotent stem cells are administered intravenously in a 1 × 10⁻¹⁶ dose. ４ A cell preparation according to any one of [1] to [4] used to administer at a rate of cell count / solids or more; a cytokine storm inhibitor comprising the cell preparation according to any one of [6], [1] to [5]; an apoptosis inhibitor comprising the cell preparation according to any one of [7], [1] to [5].

[0011] The present invention provides a cell preparation containing pluripotent stem cells that can be used to treat and / or prevent diseases such as pneumonia and pulmonary fibrosis caused by SARS-CoV-2 infection, as well as post-symptomatic symptoms such as olfactory dysfunction.

[0012] This figure shows the comparison of ACE2 and TMPRSS2 expression in Muse cells and non-Muse cells. This figure shows the weight changes of hamsters infected with SARS-CoV-2. This figure shows the flow of lung tissue collection after administering Muse cells or non-Muse cells to hamsters infected with SARS-CoV-2. This figure shows the weight changes of hamsters (control, positive control, Muse cells, non-Muse cells). Oxygen saturation (SpO) ２This figure shows the measurement results (control, positive control, Muse cells, non-Muse cells), etc. This figure shows the flow of administering Muse cells or non-Muse cells to hamsters infected with SARS-CoV-2 and collecting lung tissue, and the results of HE staining and immunostaining of the hamster lungs. This figure shows the evaluation results of inflammation suppression by Muse cell administration. This figure shows the flow of administering Muse cells or non-Muse cells to hamsters infected with SARS-CoV-2 and collecting lung tissue, and the results of TUNEL staining and HE staining of the hamster lungs. This figure shows the evaluation results of apoptosis suppression by Muse cell administration. This figure shows the flow of administering Muse cells or non-Muse cells to hamsters infected with SARS-CoV-2 and collecting lung tissue, and the results of immunostaining and HE staining of the hamster lungs. This figure shows the results of immunological analysis after administration of Muse cells. This figure shows the results of gene expression that induces macrophage migration in hamster lung cells. This figure shows the flow of administering Muse cells or non-Muse cells to hamsters infected with SARS-CoV-2 and collecting lung tissue, and the results of Sirius Red staining of the hamster lungs. This figure shows the evaluation results of fibrosis suppression by Muse cell administration. This figure shows the results of pathway analysis of Muse cells. This figure shows the flow of administering Muse cells or non-Muse cells to hamsters infected with SARS-CoV-2 and collecting olfactory bulb tissue, and the location of the olfactory bulb in hamsters. This figure shows the results of immunostaining of the hamster olfactory bulb. This figure shows the results of RNA sequencing of the hamster olfactory bulb. This figure shows the results of TUNEL staining of the hamster olfactory bulb.

[0013] The present invention is characterized by using a cell preparation containing SSEA-3-positive pluripotent stem cells (Muse cells) to treat and / or prevent diseases and post-symptomatic symptoms caused by SARS-CoV-2 infection.

[0014] 1. Applicable Diseases As described above, the cell preparation containing SSEA-3-positive pluripotent stem cells (Muse cells) of the present invention is used to treat and / or prevent diseases and post-symptomatic symptoms caused by SARS-CoV-2 infection. Therefore, the cell preparation of the present invention is characterized by being used to be administered for diseases and / or post-symptomatic symptoms caused by SARS-CoV-2 infection. Diseases caused by SARS-CoV-2 infection include pneumonia (viral pneumonia), pulmonary fibrosis, acute respiratory distress syndrome (ARDS), arrhythmia, acute heart injury, and multiple organ failure. Among these, a preferred embodiment is the use of the cell preparation of the present invention to treat and / or prevent pneumonia and / or pulmonary fibrosis. Post-COVID-19 symptoms resulting from SARS-CoV-2 infection are also known as post-COVID-19 condition, post-COVID conditions, long COVID, post-acute COVID-19 syndrome (PACS), post-acute sequence of SARS-CoV-2 infection (PASC), persistent symptoms, lingering symptoms, etc. They refer to all symptoms that persist from the acute phase or newly appear or reappear and persist during the course of COVID-19, even though infectivity has disappeared, and there is no other apparent cause. The aforementioned post-onset symptoms include respiratory symptoms such as dyspnea, shortness of breath, cough, and sputum; circulatory symptoms such as chest pain, palpitations, fatigue, edema of the extremities, coldness, syncope, and arrhythmia; olfactory and gustatory symptoms such as loss of smell and taste; neurological symptoms such as fatigue, malaise, memory impairment, decreased concentration, mood swings, brain fog, muscle weakness, headache, muscle pain, sleep disorders, dizziness when standing, and bradykinesia; psychiatric symptoms such as depression, anxiety, psychotic disorders, cognitive decline, dementia, epilepsy, and suicidal ideation; pain symptoms such as fatigue, malaise, sore throat, headache, chest pain, back pain, lower back pain, muscle pain, joint pain, and stabbing pain; and skin symptoms such as acne-like rash, livedo-like rash, vasculitic purpura, maculopapular erythematous rash, bullous rash, desquamating rash, and alopecia. In addition to post-illness symptoms, other diagnoses include interstitial pneumonia, pneumothorax, mediastinal emphysema, pulmonary thromboembolism, acute myocardial infarction, angina pectoris, cardiomyopathy, pericarditis, and Kawasaki disease in children.In other words, a preferred embodiment of the present invention is to use the cell preparation to treat and / or prevent at least one post-morbidity symptom selected from the group consisting of respiratory symptoms, circulatory symptoms, olfactory / gustatory symptoms, neurological symptoms, psychiatric symptoms, pain symptoms, and skin symptoms. More preferably, it is a preferred embodiment to use the cell preparation to treat and / or prevent at least one post-morbidity symptom selected from the group consisting of respiratory symptoms, olfactory / gustatory symptoms, neurological symptoms, and pain symptoms, and even more preferably to use it to treat and / or prevent olfactory / gustatory symptoms.

[0015] 2. Cell preparations (1) Pluripotent stem cells (Muse cells) The pluripotent stem cells used in the cell preparations of the present invention are typically cells whose existence in the human body was discovered by Mr. Izawa, one of the inventors, and which he named "Muse (Multilineage-differentiating Stress Enduring) cells." Muse cells can be obtained from bone marrow fluid, adipose tissue (Ogura, F., et al., Stem Cells Dev., Nov 20, 2013 (Epub) (published on Jan 17, 2014)), and skin tissue such as dermal connective tissue, and are also scattered throughout the connective tissue of various organs. Furthermore, these cells possess the properties of both pluripotent stem cells and mesenchymal stem cells, and are identified, for example, as double-positive for the respective cell surface markers, "SSEA-3 (Stage-specific embryonic antigen-3)" and "CD105". Therefore, Muse cells or cell populations containing Muse cells can be isolated from living tissues, for example, using these antigen markers as indicators. In addition, Muse cells are stress-tolerant and can be enriched from mesenchymal tissue or cultured mesenchymal cells by various stress stimuli. The cell preparation of the present invention can also use a cell fraction in which Muse cells have been enriched by stress stimuli. Details of the isolation method, identification method, and characteristics of Muse cells are disclosed in International Publication No. WO2011 / 007900. Furthermore, as reported by Wakao et al. (Proc. Natl. Acad. Sci. USA, Vol. 108, pp. 9875-9880, 2011), when mesenchymal cells are cultured from bone marrow, skin, etc., and used as a population of Muse cells, it has been found that all SSEA-3 positive cells are CD105 positive cells. Therefore, in the cell preparation of the present invention, when Muse cells are isolated from living mesenchymal tissue or cultured mesenchymal stem cells, Muse cells can simply be purified and used using SSEA-3 as an antigen marker. In this specification, pluripotent stem cells (Muse cells) or cell populations containing Muse cells isolated from living mesenchymal tissue or cultured mesenchymal tissue using SSEA-3 as an antigen marker may be simply referred to as "SSEA-3 positive cells".Furthermore, in this specification, "non-Muse cells" refers to cells contained in living mesenchymal tissue or cultured mesenchymal tissue, other than "SSEA-3 positive cells." As shown in the examples described later, Muse cells can be isolated from human bone marrow mesenchymal stem cells (BM-MSCs) by FACS sorting targeting SSEA-3 positivity. Non-Muse cells can be obtained as the remainder of human bone marrow mesenchymal stem cells (BM-MSCs) other than the isolated Muse cells. Muse cells and non-Muse cells can also be obtained in accordance with the method described in International Publication No. WO2011 / 007900.

[0016] Muse cells or cell populations containing Muse cells can be isolated from living tissue (e.g., mesenchymal tissue) using either an antibody against the cell surface marker SSEA-3 alone, or antibodies against SSEA-3 and CD105, respectively. Here, "living organism" refers to a mammalian organism. In this invention, the organism does not include embryos at developmental stages prior to the blastula stage, such as fertilized eggs or blastulas, but it does include embryos at developmental stages from the blastula stage onward, including fetuses and blastulae. Examples of mammals, though not limited to them, include humans, primates such as monkeys, rodents such as mice, rats, rabbits, and guinea pigs, cats, dogs, sheep, pigs, cattle, horses, donkeys, goats, and ferrets. The Muse cells used in the cell preparation of this invention are clearly distinguished from embryonic stem cells (ES cells) and iPS cells in that they are isolated directly from living tissue using markers. Furthermore, "mesenchymal tissue" refers to tissues such as bone, synovial membrane, fat, blood, bone marrow, skeletal muscle, dermis, ligaments, tendons, dental pulp, umbilical cord, umbilical cord blood, and tissues present in various organs. For example, Muse cells can be obtained from bone marrow, skin, or adipose tissue. For example, it is preferable to collect mesenchymal tissue from a living organism, isolate Muse cells from this tissue, and utilize them. Alternatively, Muse cells may be isolated from cultured mesenchymal cells such as fibroblasts or bone marrow mesenchymal stem cells using the above-mentioned isolation method. In the cell preparation of the present invention, the Muse cells used may be autologous or allogeneic to the recipient.

[0017] As described above, Muse cells or cell populations containing Muse cells can be isolated from living tissue using indicators such as SSEA-3 positivity and double positivity for SSEA-3 and CD105. However, adult human skin is known to contain various types of stem cells and progenitor cells. Muse cells, however, are not the same as these cells. Examples of such stem cells and progenitor cells include skin-derived progenitor cells (SKP), neural crest stem cells (NCSC), melanoblast (MB), perivascular cells (PC), endothelial progenitor cells (EP), and adipose-derived stem cells (ADSC). Muse cells can be isolated using the "non-expression" of markers specific to these cells as an indicator. More specifically, Muse cells can be isolated based on the non-expression of at least one of 11 markers selected from the group consisting of CD34 (marker for EP and ADSC), CD117 (c-kit) (marker for MB), CD146 (marker for PC and ADSC), CD271 (NGFR) (marker for NCSC), NG2 (marker for PC), vWF factor (von Willebrand factor) (marker for EP), Sox10 (marker for NCSC), Snai1 (marker for SKP), Slug (marker for SKP), Tyrp1 (marker for MB), and Dct (marker for MB). For example, non-expression of 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 markers can be used as an indicator. For example, but not limited to, the absence of CD117 and CD146 can be used as indicators for separation; further, the absence of CD117, CD146, NG2, CD34, vWF, and CD271 can be used as indicators for separation; and further, the absence of the above 11 markers can be used as indicators for separation.

[0018] Furthermore, Muse cells having the above characteristics used in the cell preparation of the present invention may have at least one property selected from the group consisting of: (i) low or absent telomerase activity; (ii) ability to differentiate into cells of any of the three germ layers; (iii) not exhibiting neoplastic growth; and (iv) self-renewal ability. In one aspect of the present invention, Muse cells used in the cell preparation of the present invention have all of the above properties. Here, with respect to (i) above, "low or absent telomerase activity" means, for example, that when telomerase activity is detected using the TRAPEZE XL telomerase detection kit (Millipore), it is low or undetectable. "Low" telomerase activity means, for example, that it has telomerase activity at the same level as human fibroblasts, which are somatic cells, or that it has telomerase activity of 1 / 5 or less, preferably 1 / 10 or less, compared to Hela cells. Regarding (ii) above, Muse cells have the ability to differentiate into three germ layers (endoderm, mesoderm, and ectoderm) in vitro and in vivo. For example, they can differentiate into hepatocytes, nerve cells, skeletal muscle cells, smooth muscle cells, osteocytes, adipocytes, etc., by induction culture in vitro. They may also exhibit the ability to differentiate into three germ layers when transplanted into the testes in vivo. Furthermore, when transplanted into a living organism by intravenous injection, they have the ability to migrate and engraft in damaged organs (heart, skin, spinal cord, liver, muscle, etc.) and differentiate into cells appropriate to the tissue. Regarding (iii) above, Muse cells proliferate in suspension culture at a rate of approximately 1.3 days, but in suspension culture, they proliferate from a single cell, form embryoid body-like cell aggregates, and proliferation stops after about 14 days. However, when these embryoid body-like cell aggregates are taken to adherent culture, cell proliferation starts again, and the cells that have proliferated from the cell aggregates spread. Furthermore, when transplanted into the testes, they have the property of not becoming cancerous for at least six months. Also, regarding (iv) above, Muse cells have the ability to self-renew (self-replicate).Here, "self-renewal" refers to the process where, by culturing a single Muse cell in suspension culture, differentiation of cells within an embryoid body-like cell aggregate into three germ layer cells can be observed. Simultaneously, by returning the cells from the embryoid body-like cell aggregate to suspension culture as a single cell, the next generation of embryoid body-like cell aggregates can be formed, and from these, three germ layer differentiation and embryoid body-like cell aggregates in suspension culture can be observed again. Self-renewal can be achieved by repeating the cycle once or multiple times.

[0019] Furthermore, the cell fraction containing Muse cells used in the cell preparation of the present invention may be a cell fraction enriched with SSEA-3-positive and CD105-positive pluripotent stem cells having at least one, preferably all, of the following properties, obtained by a method that includes applying an external stress stimulus to mesenchymal tissue or cultured mesenchymal cells in a living organism, killing cells other than those resistant to the external stress, and collecting the surviving cells: (i) SSEA-3 positive; (ii) CD105 positive; (iii) low or absent telomerase activity; (iv) ability to differentiate into three germ layers; (v) no neoplastic growth; and (vi) self-renewal ability.

[0020] The above external stresses may be any or a combination of the following: protease treatment, culture at low oxygen concentration, culture under low phosphate conditions, culture at low serum concentration, culture under nutrient-poor conditions, culture under exposure to heat shock, culture at low temperature, freezing treatment, culture in the presence of harmful substances, culture in the presence of reactive oxygen species, culture under mechanical stimulation, culture under shaking treatment, culture under pressure treatment, or physical shock. For example, the protease treatment time is preferably 0.5 to 36 hours in total to apply external stress to the cells. The protease concentration may be any concentration used when detaching cells adhered to a culture vessel, separating cell aggregates into single cells, or recovering single cells from tissue. The protease is preferably serine protease, aspartate protease, cysteine ​​protease, metalloprotease, glutamate protease, or N-terminal threonine protease. Furthermore, it is preferable that the protease is trypsin, collagenase, or dispase.

[0021] (2) Preparation and Use of Cell Preparations The cell preparations of the present invention can be obtained by suspending the Muse cells or cell populations containing Muse cells obtained in (1) above in physiological saline or an appropriate buffer (e.g., phosphate-buffered saline), although this is not limited to these preparations. In this case, if the number of Muse cells isolated from autologous or allogeneic tissue is small, the cells may be cultured before cell transplantation and grown until a predetermined number of cells is obtained. As has already been reported (International Publication No. WO2011 / 007900), Muse cells do not become cancerous, so even if cells recovered from living tissue are included in an undifferentiated state, there is little concern about cancer and they are safe. Furthermore, the culture of recovered Muse cells can be carried out in a normal growth medium (e.g., α-minimum essential medium (α-MEM) containing 10% calf serum), although this is not particularly limited. For more details, please refer to International Publication No. WO2011 / 007900 mentioned above. In culturing and expanding Muse cells, appropriate culture media, additives (e.g., antibiotics, serum), etc., can be selected to prepare a solution containing Muse cells at a predetermined concentration. When administering the cell preparation of the present invention to human subjects, bone marrow fluid can be collected from the iliac bone, and for example, bone marrow mesenchymal stem cells can be cultured as adherent cells from the bone marrow fluid until an effective therapeutic dose of Muse cells is obtained. Then, the Muse cells can be separated using the SSEA-3 antigen marker as an indicator, and autologous or allogeneic Muse cells can be prepared as a cell preparation. Alternatively, for example, bone marrow mesenchymal stem cells obtained from bone marrow fluid can be cultured under external stress conditions to expand and concentrate Muse cells until an effective therapeutic dose is obtained, and then autologous or allogeneic Muse cells can be prepared as a cell preparation.

[0022] Also, in the use of Muse cells in cell preparations, dimethyl sulfoxide (DMSO), serum albumin, etc. may be contained in the cell preparation to protect the cells, and antibiotics, etc. may be contained in the cell preparation to prevent contamination and growth of bacteria. Furthermore, other pharmaceutically acceptable components (for example, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.) may be contained in the cell preparation. Those skilled in the art can add these factors and drugs to the cell preparation at appropriate concentrations. That is, the cell preparation of the present invention can be used as a pharmaceutical composition. Therefore, in this specification, a cell preparation containing SSEA-3 positive pluripotent stem cells (Muse cells) may be read as a pharmaceutical composition containing SSEA-3 positive pluripotent stem cells (Muse cells).

[0023] The number of Muse cells contained in the cell preparation prepared above can be appropriately adjusted in consideration of the sex, age, weight, condition of the affected part, condition of the cells to be used, etc. of the subject so as to obtain the desired effect. The subject individuals include, but are not limited to, mammals such as humans. Also, the cell preparation of the present invention may be administered a plurality of times (for example, 2 to 10 times) at appropriate intervals (for example, twice a day, once a day, twice a week, once a week, once every two weeks, once a month, once every two months, once every three months, once every six months) until the desired therapeutic effect is obtained. Therefore, depending on the condition of the subject, as an effective amount to be administered, for example, 1×10 ３ cells / solid to 1×10 １０ cells / solid, and the dosage for 1 to 10 administrations is preferably 5×10 ３ cells / solid to 1×10 ９ cells / solid is more preferable, and 1×10 ４ cells / solid to 1×10 ８ cells / solid is even more preferable, and 3×10 ４ cells / solid to 1×10 ７ cells / solid is particularly preferable. The total dosage for one individual is not limited, but 1×10 ３ cells / solid to 1×10 １１ cells / solid is preferable, and 1×10 ４ cells / solid to 1×10１０ A cell count / solid is more preferably 3 × 10 ４ Number of cells / solid ~ 1 x 10 ９ A higher cell-to-solid ratio is preferable.

[0024] The cell preparation of the present invention is not particularly limited in terms of the site or form of administration. It may be administered locally by injection or other methods to the disease site or its vicinity, or it may be administered intravascularly by injection or other methods. The type of blood vessel (vein and artery) into which it is administered is not particularly limited and can be appropriately selected depending on the disease, but intravenous injection is a preferred embodiment, and multiple intravenous injections are a more preferred embodiment.

[0025] As shown in the examples described later, Muse cells significantly suppressed major cytokines involved in inflammation and significantly suppressed apoptosis caused by SARS-CoV-2. Therefore, a cytokine storm inhibitor containing the cell preparation of the present invention is a preferred embodiment, and an apoptosis inhibitor containing the cell preparation of the present invention is also a preferred embodiment.

[0026] The present invention will be described in more detail below using examples. In these examples, the Syrian hamster (hereinafter sometimes abbreviated as "hamster") used was a 5-week-old female purchased from Nippon SLC Co., Ltd. SARS-CoV-2 was provided by the National Institute of Infectious Diseases and was grown by infecting cells with VeroE6 / TMPRSS2.

[0027] Example 1 (Preparation of Muse and Non-Muse Cells) Muse cells were isolated from human bone marrow mesenchymal stem cells (BM-MSCs) by FACS sorting targeting SSEA-3 positivity. Non-Muse cells were obtained as the remainder of human bone marrow mesenchymal stem cells (BM-MSCs) other than the isolated Muse cells. Muse cells and non-Muse cells were maintained in low-glucose DMEM stem cell medium supplemented with 10% fetus bovine serum (FBS), 0.1 mg / mL kanamycin, and 1 ng / mL basic fibroblast growth factor (FGF-2). Muse cells can also be obtained according to the method described in International Publication No. WO2011 / 007900.

[0028] Example 2 (Administration of Muse cells and non-Muse cells) Five-week-old hamsters were acclimatized for two weeks in a P3 level infected animal laboratory, and 0.2 mL of SARS-CoV-2 was administered from the nasal cavity of seven-week-old hamsters. 5.5 TCID 50 The hamsters were infected with a solution prepared to contain 0.1 mL of the substance. Two days later, 7.0 × 10¹⁴ oz was extracted from the sublingual vein of the hamsters. 4 A number of Muse cells (n=3) or non-Muse cells (n=4) were intravenously administered to hamsters, and they were euthanized eight days after SARS-CoV-2 nasal infection. Hamsters infected with SARS-CoV-2 alone (infect; n=4) were used as positive controls, and hamsters administered 0.2 mL of saline solution into their nasal cavity (cont; n=4) were used as negative controls. Immediately after euthanasia, the lungs and olfactory bulbs (OB), which are part of the brain responsible for the sense of smell, were removed. Half of the lung and one olfactory bulb (there are two olfactory bulbs, one on each side) were fixed by immersion in 4% paraformaldehyde and subjected to HE staining and various other immunostaining methods. RNA was extracted from the remaining half of the lung and the other olfactory bulb and analyzed by RNA sequencing.

[0029] Example 3 (Confirmation of ACE2 and TMPRSS2 Expression) COVID-19 infection does not occur unless the SARS-CoV-2 spike protein is cleaved by the TMPRSS2 enzyme present in the human cell membrane after binding to the angiotensin-converting enzyme 2 (ACE2), which is the receptor for SARS-CoV-2. First, we checked whether Muse cells and non-Muse cells possessed ACE2 and TMPRSS2 by PCR. As a result, ACE2 and TMPRSS2 were expressed in Vero cells, which are known to possess these enzymes, but neither ACE2 nor TMPRSS2 were expressed in Muse cells or non-Muse cells (see Figure 1). It is already known that mouse-derived NIH / 3T3 does not get infected with SARS-CoV-2. This is because ACE2 and TMPRSS2 are not expressed in these mice. Thus, it was confirmed in advance that when human Muse cells or non-Muse cells were intravenously administered to hamsters, these cells did not become infected with SARS-CoV-2 and were unaffected. Furthermore, since Muse cells and non-Muse cells express Human Leukocyte Antigen-G (HLA-G), which is highly expressed in the umbilical cord to prevent rejection by the immune response of the mother and fetus, Muse cells and non-Muse cells are not rejected for at least two weeks when transplanted into other non-human hosts. This is because Muse cells have mechanisms to evade immune attack through immunosuppressive mechanisms and immune tolerance functions. Therefore, no immunosuppressants were administered when administering human Muse cells or non-Muse cells to hamsters. This also suggests that HLA-G expressed in Muse cells and non-Muse cells has an immune tolerance function.

[0030] Example 4 (Weight changes in hamsters infected with SARS-CoV-2) SARS-CoV-2 was administered intranasally to hamsters, and their weight was measured daily. As shown in Figure 2, when comparing the group infected with SARS-CoV-2 (Infect) with the control group that was not infected (Cont), the weight of the Infected group decreased significantly for 6-7 days after infection. In the control group, the weight gradually increased.

[0031] Example 5 (Effect of Muse cell administration on reducing weight loss, etc.) Figures 4 and 5 show the results of measuring the body weight and oxygen saturation (SpO2) of hamsters. As a result, the body weight of the Muse cell intravenous injection group (Muse) was significantly increased compared to the positive control (Infect) and the non-Muse cell intravenous injection group (non-Muse) (Figure 4, A-D). The hamsters were euthanized on the 8th day after SARS-CoV-2 infection, their lungs were removed, immersed in 4% paraformaldehyde, and stained with hematoxylin and eosin (HE) to measure the area of ​​normal alveoli (air space). As a result, the Muse cell intravenous injection group had a significantly increased air space compared to the positive control (Infect) and the non-Muse cell intravenous injection group (non-Muse) (Figure 5, A-B). Furthermore, after SARS-CoV-2 infection, oxygen saturation (SpO2) was measured in hamsters using the inhalation anesthetic isoflurane by attaching a measuring device to the hamster's leg (Figure 5, C). As a result, oxygen saturation (SpO2) was also significantly increased in the Muse cell administration group compared to the positive control (Infect) and non-Muse groups (Figure 5, D). This indicates that Muse cell administration has the effect of mitigating the effects of SARS-CoV-2 nasal infection, such as weight loss.

[0032] Example 6 (Evaluation of inflammation suppression by Muse cell administration) Hamsters were infected with SARS-CoV-2, and two days later, Muse cells or non-Muse cells were intravenously injected. Eight days after SARS-CoV-2 infection, the hamsters were euthanized and lung tissue was collected (Figure 6, A). Neutrophil accumulation in the hamster lungs was analyzed by HE staining, and Muse cells showed significantly reduced neutrophil accumulation compared to positive controls (Infect) and non-Muse cells (Figure 6, B1-B2; Figure 7, A). In addition, the hamster lungs were immunostained using MPO (Myeloperoxidase) antibody secreted from neutrophils (Figure 6, B3-B4). As a result, in the lungs of hamsters administered with Muse cells, the number of MPO-positive cells per unit area was significantly reduced compared to positive controls (Infect) and non-Muse cells (Figure 7, B). Furthermore, RNA sequencing results showed that Muse cells significantly reduced inflammation-related RNA compared to positive controls (Infect) and non-Muse cells (Figure 7, C; in the heatmap, yellow indicates strongly expressed genes, and stronger blue indicates stronger suppression of gene expression). These results suggest that Muse cells suppressed the migration of neutrophils that gather due to inflammation, thereby strongly suppressing inflammation.

[0033] Example 7 (Evaluation of Apoptosis Suppression by Muse Cell Administration) Lung tissue was collected in the same manner as in Example 6 (Figure 8, A). Hamster lungs were stained with TUNEL to examine apoptosis (cell death) (Figure 8, B1-B3; B4 and B5 were stained with HE). As a result, Muse cells significantly suppressed apoptosis compared to the positive control group (Infect) (Figure 9, A). No significant difference was observed between the positive control group (Infect) and non-Muse cells, indicating that Muse cells tend to have a greater anti-apoptotic effect than non-Muse cells (Figure 9, A). RNA sequencing results showed that Muse cells significantly suppressed the expression of apoptosis-related genes in hamster lung cells compared to the positive control group (Infect) and non-Muse cells (Figure 9, B).

[0034] Example 8 (Immunological analysis by administration of Muse cells) The lung tissue was collected in the same manner as in Example 6 (Figure 10, A). The lungs of hamsters were immunostained with an anti-SARS-CoV-2 antibody (Figure 10, B1 - B3; B4 and B5 are HE staining). As a result, in the lungs of hamsters administered with Muse cells, the proportion of cells that were significantly SARS-CoV-2 positive was lower compared to the positive control (Infect), but no significant difference was observed compared to non-Muse cells (Figure 11, A). As a result of examining the RNA related to immunity from the results of RNAseq, Muse cells significantly increased the expression of genes (Figure 11, B; C1qc, C3, Ca4, C5, Csf1) that migrate macrophages in hamster lung cells compared to the positive control (Infect) and non-Muse cells (Figure 12).

[0035] Example 9 (Evaluation of fibrosis fibrosisfib of fibrosis suppression by administration of Muse cells) The lung tissue was collected in the same manner as in Example 6 (Figure 13, A). The lungs of hamsters were stained with Sirius red to examine lung fibrosis (Figure 13, B1 - B2). As a result, the area that became Sirius red positive was significantly decreased in Muse cells compared to the positive control (infect), and lung fibrosis was suppressed (Figure 14, A). Although no significant difference was found between the positive control (Infect) and non-Muse cells, or between non-Muse cells and Muse cells, it was revealed that Muse cells tended to suppress fibrosis compared to non-Muse cells (Figure 14, A). The RNA gene groups related to fibrosis were examined by RNAseq. As a result, it was suggested that Muse cells significantly suppressed the expression of gene groups involved in fibrosis in hamster lung cells compared to the positive control (Infect) and non-Muse cells (Figure 14, B).

[0036] Example 10 (Pathway Analysis of Muse Cells) From the RNA-seq data of hamster lungs, only RNAs that were significantly increased or decreased in Muse cells compared to non-Muse cells were input into the analysis software Ingenuity Pathway Analysis (IPA) to analyze pathways (Figure 15). If the levels were significantly lower in Muse cells compared to non-Muse cells, they were displayed in blue, and if they were increased, they were displayed in pink. As a result, Muse cells significantly suppressed the major cytokines involved in inflammation, IL-6, TNFα, and IL-1β, compared to non-Muse cells. In COVID-19, it has been reported that a runaway surge of inflammatory cytokines called a cytokine storm leads to the severity of COVID-19 pneumonia. As in this example, if the cytokine storm is suppressed by intravenous injection or infusion of Muse cells, it is suggested that pneumonia caused by SARS-CoV-2 infection will not become severe. Furthermore, it was found that Muse cells suppressed the expression of Colony-Stimulating Factor (CSF) in hamster lungs. It is known that CSF levels surge in the serum of patients with severe COVID-19, and it is hoped that suppressing CSF expression in hamster lungs using Muse cells will prevent COVID-19 from becoming severe.

[0037] Example 11 (Inhibitory effect of Muse cell administration on the olfactory bulb) Hamsters were infected with SARS-CoV-2, and two days later, Muse cells or non-Muse cells were intravenously injected. Eight days after SARS-CoV-2 infection, the hamsters were euthanized and olfactory bulb tissue was collected (Figure 16, A). The arrow in Figure 16B indicates the olfactory bulb. The hamster olfactory bulbs were immunostained using anti-SARS-CoV-2 antibody (Figure 17, B). As a result, the area positive for anti-SARS-CoV-2 antibody was significantly reduced in both Muse cells and non-Muse cells compared to the positive control (Infect) (Figure 17, A). Similarly, RNA sequencing results showed an increase in SARS-CoV-2 genes in the positive control (Infect), but both Muse cells and non-Muse cells were dramatically reduced compared to the positive control (Infect) (Figure 18). The hamster olfactory bulbs were stained with TUNEL to examine apoptosis (programmed cell death) (Figure 19, B). As a result, the area of ​​TUNEL staining positivity was significantly reduced in Muse cells compared to the positive control (Infect) (p<0.001). Non-Muse cells also showed a similar cell death suppression effect compared to the positive control (Infect) (p<0.01), but the statistical significance was higher between Muse cells and the positive control group (p<0.001). Therefore, although there was no significant difference between Muse cells and non-Muse cells, it was suggested that Muse cells tended to have a greater cell death suppression effect (Figure 19, A). Thus, Muse cells significantly suppressed SARS-CoV-2 proliferation in the olfactory bulb compared to non-Muse cells (Figure 18), and furthermore, they significantly suppressed SARS-CoV-2-induced apoptosis more than the positive control (Infect) and non-Muse cells.

[0038] The following was revealed from the results of the above-mentioned examples: Intravenous administration of Muse cells to hamsters improved pneumonia caused by SARS-CoV-2 infection and suppressed cell death in the olfactory bulb more effectively than intravenous administration of non-Muse cells. The effects were as follows: (1) Muse cells suppressed alveolar damage caused by SARS-CoV-2, and as a result, suppressed the decrease in SpO2 without causing weight loss or reduction in alveolar area. (2) The mechanism by which Muse cells suppress alveolar damage caused by SARS-CoV-2 was found to be the effect of suppressing cytokine storms by suppressing inflammation. (3) Administration of Muse cells showed anti-apoptotic and anti-fibrotic effects in lung tissue, and is expected to be effective against post-symptomatic symptoms known as Long COVID-19. The Ministry of Health, Labour and Welfare has expressed the following view regarding pulmonary fibrosis, a post-symptomatic symptom caused by SARS-CoV-2 infection. "Regarding imaging, a meta-analysis of 46 papers on viral pneumonia caused by SARS-CoV-2 reported that inflammatory shadows (such as ground-glass opacities) were observed in 50% of patients up to 12 months after hospitalization, and significantly decreased over time, while fibrotic findings were observed in 29% of patients, and although they decreased over time, the decrease was not statistically significant (Ministry of Health, Labour and Welfare supervised, Management of Post-Illness Symptoms, 3.0 edition, page 23)." (4) It was found that Muse cells and non-Muse cells have the effect of eliminating SARS-CoV-2 itself through the immune system. Macrophages were thought to be involved in this. (5) Furthermore, both Muse cells and non-Muse cells significantly suppressed the proliferation of SARS-CoV-2 in the olfactory bulb compared to the positive control (Infect), and apoptosis of the olfactory bulb due to SARS-CoV-2 infection was also significantly suppressed in Muse cells compared to the positive control (Infect) and non-Muse cells. This apoptosis of the olfactory bulb due to SARS-CoV-2 infection was thought to be significantly involved in post-symptomatic symptoms of SARS-CoV-2 infection. The Ministry of Health, Labour and Welfare has issued the following statement regarding olfactory dysfunction as a post-symptomatic symptom of SARS-CoV-2 infection."On the other hand, regarding cases where the impairment persists for a long period, although the mechanism is not fully understood, it is thought that the impairment is not limited to supporting cells but extends to olfactory nerve cells, resulting in persistent olfactory dysfunction that is reported as a post-symptom (Ministry of Health, Labour and Welfare supervised, Management of Post-Symptoms, 3.0 edition, page 31)." Muse cells are also expected to be effective for olfactory dysfunction, a post-symptom known as Long COVID-19.

Claims

1. A cell preparation comprising SSEA-3-positive pluripotent stem cells derived from living mesenchymal tissue or cultured mesenchymal cells, characterized in that it is used to treat diseases caused by SARS-CoV-2 infection and / or post-symptomatic symptoms.

2. The cell preparation according to claim 1, wherein the pluripotent stem cells are used to treat and / or prevent diseases and / or post-symptomatic symptoms caused by SARS-CoV-2 infection.

3. The cell preparation according to claim 1 or 2, wherein the disease is pneumonia and / or pulmonary fibrosis.

4. The cell preparation according to claim 1 or 2, wherein the post-illness symptom is olfactory dysfunction.

5. The pluripotent stem cells are administered intravenously in a dose of 1 x 10⁶ ４ A cell preparation according to claim 1 or 2, used to administer a number of cells / solids equal to or greater than the number of cells / solids.

6. A cytokine storm inhibitor comprising the cell preparation according to claim 1 or 2.

7. An apoptosis inhibitor comprising the cell preparation according to claim 1 or 2.

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