Tissue regeneration with in vitro activated effector cells

Abstract

A composition is provided that includes a) activated peripheral blood mononuclear cells (PBMCs) and b) a co-culture of stem or progenitor cells, the composition being useful for regenerating tissue in a subject in need of tissue regeneration and for forming tissue in vitro.

Description

[Technical field]

[0001] [Related Applications] This application claims priority to U.S. Provisional Patent Application No. 63 / 279,316, filed November 15, 2021, the contents of which are incorporated herein by reference. [Background technology]

[0002] Knee trauma occurs primarily in the younger population, with over 50% occurring in individuals between 15 and 44 years of age. Post-traumatic osteoarthritis (PTOA) begins early, even when relatively asymptomatic. Outcome studies of PTOA have shown radiographic evidence of knee osteoarthritis (KOA) in 12-20% of patients after 5 years. Treatment of KOA involves a lifelong combination of exercise, lifestyle modifications, corticosteroid injections, pain medications, and, in severe cases, prosthetic knee replacement surgery. See Sharma, “Osteoarthritis of the Knee,” The New England Journal of Medicine, January 7, 2021. Non-surgical treatments can only provide pain relief and do not slow the progression of KOA.

[0003] Cell therapy with administration of mesenchymal stem cells, such as adipose tissue-derived stromal cells, has been investigated for osteoarthritis treatment and may have anti-inflammatory properties. However, some studies have shown benefit from the treatment, while others have not. See, for example, van den Bosch, “Review: Osteoarthritis year in review 2020: biology,” Osteoarthritis and Cartilage 29(2021)143-150; Zhang et al., “Review: Progress in the use of mesenchymal stromal cells for osteoarthritis treatment,” Cytotherapy 23(2021)459-470; Soltani et al., “Safety and efficacy of allogenic placental mesenchymal stem cells for treating knee osteoarthritis: a pilot study,” Cytotherapy 21(2019)54-63. This treatment also provides only short-term benefits, mainly through reduction in pain. Little or no structural regeneration is achieved, leading to later disease progression.

[0004] Jaewoo Pak's US Patent Application Publication No. 2012 / 0171169 relates to a composition for the treatment of bone disease, comprising adipose tissue-derived stem cells, platelet-rich plasma, calcium chloride and hyaluronic acid, and in some embodiments further comprising dexamethasone for the treatment of cartilage disease. However, there is no comparative example showing that this stem cell treatment results in improved bone or cartilage cell recovery over other methods of stem cell administration known in the art.

[0005] Better treatment options for tissue regeneration, such as for PTOA, are needed. Summary of the Invention

[0006] According to some embodiments herein, there is provided a composition comprising a) activated peripheral blood mononuclear cells (PBMCs) and b) a co-culture of stem cells or progenitor cells, wherein the activated PBMCs and the stem cells or progenitor cells are present in the composition at a ratio of activated PBMCs:stem cells or progenitor cells of 6:1, 5:1, 4.5:1 or 4:1 to 3:1, 2.5:1 or 2:1. In some embodiments, the activated PBMCs and the stem cells or progenitor cells are present in the composition at a ratio of activated PBMCs:stem cells or progenitor cells of 4.5:1 or 4:1 to 2.5:1 or 2:1.

[0007] In some embodiments, the stem or progenitor cells are placenta-derived progenitor cells, hi some embodiments, the stem or progenitor cells are mesenchymal stem cells.

[0008] In some embodiments, the activated PBMCs are from a different subject than the stem or progenitor cells. In some embodiments, the activated PBMCs and the stem or progenitor cells are both from the same subject.

[0009] In some embodiments, the activated PBMCs are activated with an antigen derived from a tissue selected from the group consisting of cartilage, bone, muscle, nerve and brain tissue.

[0010] In some embodiments, the activated PBMCs are activated with an antigen derived from a tissue selected from the group consisting of cartilage and bone tissue.

[0011] In some embodiments, the antigen is an enzymatic hydrolysate of the tissue.

[0012] In some embodiments, the compositions are formulated for injection or infusion into damaged or diseased tissue.

[0013] In some embodiments, the activated PBMCs and the stem or progenitor cells are co-cultured for less than 96, 72, or 48 hours (eg, about 24 hours) prior to use.

[0014] In some embodiments, the composition further comprises a hydrogel carrier and / or hyaluronic acid.

[0015] Also provided is a method of regenerating tissue in a subject in need thereof comprising administering to the subject a therapeutically effective amount of a composition taught herein, or the use of a composition taught herein to regenerate tissue in a subject in need thereof.

[0016] In some embodiments, the administration occurs about 4 or 5 days to about 11 or 12 days after tissue injury, such as about 6 days to about 10 days after tissue injury, In some embodiments, the administration occurs about 1, 2, 3, or 4 weeks after tissue injury.

[0017] In some embodiments, the activated PBMCs are obtained by extraction from the subject's or donor's whole blood, apheresis, or buffy coat.

[0018] In some embodiments, the PBMCs are autologous with respect to the subject in need of regeneration of the tissue, and the stem or progenitor cells are allogeneic with respect to the subject in need of regeneration of the tissue.

[0019] In some embodiments, the composition is administered locally to the site of a wound or disease in a subject in need of regeneration of said tissue, hi some embodiments, the site of the wound or disease is the knee of the subject.

[0020] In some embodiments, the composition is administered systemically and the PBMCs and / or stem or progenitor cells migrate to the site of the wound or disease in the subject in need of regeneration of the tissue, hi some embodiments, the site of the wound or disease is the knee of the subject.

[0021] Further provided is a method of forming tissue in vitro comprising providing a tissue scaffold and seeding the scaffold with stem or progenitor cells and activated PBMCs, whereby the stem or progenitor cells differentiate into tissue in vitro.

[0022] In some embodiments, the activated PBMCs are activated with an antigen derived from a tissue selected from the group consisting of cartilage, bone, muscle, nerve and brain tissue.

[0023] In some embodiments, the stem or progenitor cells and activated PBMCs are seeded together onto the tissue scaffold as a co-culture.

[0024] In some embodiments, the stem or progenitor cells and activated PBMCs are seeded separately onto the tissue scaffold. [Brief description of the drawings]

[0025] [Figure 1] FIG. 1 shows that in vitro mechanistic evaluation of cartilage-activated effector cells (ECs) confirmed stimulation of macrophages and lymphocytes as indicated by increased CD68, CD69 and CD25 compared to primary peripheral blood mononuclear cell populations (MNCs). [Diagram 2] FIG. 1 shows that measurements of gene expression in chondrogenically activated ECs showed increased expression of the pro-regenerative markers TNF-alpha and IL10, along with increased expression of the chondrogenic differentiation-inducing factors COMP and BMP2, compared to primary MNC populations. [Diagram 3] FIG. 1 shows that upon 24-hour co-culture of ECs with placenta-derived progenitor cells (PLC), the onset of chondrocyte differentiation was confirmed by the transient upregulation of the transcription factor, cartilage matrix protein and synovial lubricant Prg4. [Figure 4]When the cell populations were injected into three models of functional injury healing and then pathologically assessed 12 weeks after injury creation, the co-cultured cell treatments, but not the other cell treatments with each of the cell populations alone, induced functional regeneration, as confirmed by (1) the apex between the subchondral bone and the articular unit, (2) glycosaminoglycan-rich subchondral articular cartilage, and (3) the restoration of an articular chondrocyte layer secreting lubricin, a qualitative marker of functional articular cartilage. [Diagram 5] FIG. 1 shows that administration of placental cells alone produced an anti-inflammatory effect and partial regeneration, but did not induce the rapid tissue repair and functional regeneration as did the co-cultured composition. [Figure 6] FIG. 1 shows that patients enrolled in a compassionate use trial reported significant improvements in pain, functionality and quality of life 180 days after treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0026] The present invention will now be described more fully hereinafter with reference to the accompanying drawings, in which embodiments of the invention are shown. However, the present invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0027] The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the present invention. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms unless the context clearly indicates otherwise. It will be further understood that the terms "comprises" or "comprising", as used herein, specify the presence of stated features, integers, steps, operations, elements, components and / or groups or combinations thereof, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups or combinations thereof.

[0028] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. It will be further understood that terms such as those defined in commonly used dictionaries should be interpreted to have a meaning that is not inconsistent with their meaning in the context of the present specification and claims, and should not be interpreted in an idealized or overly formal sense unless expressly defined herein. Well-known functions or configurations may not be described in detail for brevity and / or clarity.

[0029] All publications, patent applications, patents, and other references mentioned herein are incorporated by reference to the extent they do not conflict with the disclosure set forth herein. In the case of conflicting terms, the present specification controls.

[0030] As used herein, "and / or" refers to and includes any and all possible combinations of one or more of the associated listed items, and when interpreted alternatively ("or"), no combinations.

[0031] Unless otherwise indicated by the context, it is specifically intended that the various features of the present invention described herein can be used in any combination.Furthermore, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features described herein can be excluded or omitted.For example, if the present specification states that a composition comprises components A, B and C, it is specifically intended that any of A, B, or C or combinations thereof can be omitted and negated.

[0032] As used herein, the term "about" when referring to a measurable value, such as an amount or concentration, is intended to encompass a variation of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value and the specified value. For example, "about X", where X is a measurable value, is intended to include X and ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. Furthermore, ranges provided herein for measurable values ​​can include any other ranges and / or individual values ​​therein.

[0033] The "cells" used in the present invention are generally animal cells, particularly mammalian and primate cells, including, but not limited to, human, dog, cat, rabbit, monkey, chimpanzee, cow, pig, or goat. The cells may be stem or progenitor cells, or may be at least partially differentiated into specific cell or tissue types, such as cartilage, bone, muscle (smooth, skeletal, cardiac), nerve (central, peripheral), brain tissue, etc. The cells may be obtained from established cultures, donors, biopsies, or combinations thereof, as known in the art.

[0034] In some embodiments, the cells may be in culture expanded prior to use. "Expanding" as used herein refers to an increase in the number of viable cells, and may be accomplished, for example, by growing the cells through one or more cell cycles in which at least a portion of the cells divide to produce additional cells.

[0035] A "subject," as used herein, is generally a human subject, although aspects of the invention may be practiced with other animal subjects, particularly mammalian subjects (e.g., dogs, cats, horses, goats, sheep), for research or veterinary purposes. Subjects may be male or female of any age, including infants, juveniles, adolescents, adults, and geriatrics.

[0036] "Treating," as used herein, refers to any type of treatment that provides a benefit to a subject, including, but not limited to, delaying the onset or reducing the severity of at least one symptom associated with tissue injury or disease, such as osteoarthritis, improving tissue or joint function, etc. Similarly, a "therapeutically effective" amount is an amount sufficient to treat and provide such benefit.

[0037] A "medium" herein can be any natural or artificial growth medium (typically an aqueous liquid) that supports cells used in the practice of the present invention. Examples include, but are not limited to, basal medium or minimal essential medium (MEM), or variations thereof, such as Eagle's minimal essential medium (EMEM) and Dulbecco's modified Eagle's medium (DMEM). In some embodiments, the growth medium includes a pH color indicator (e.g., phenol red).

[0038] As known in the art, stem cells and progenitor cells are cells that can differentiate into one or more specific types of tissue cells.Stem cells can generally differentiate into many more types of cells than progenitor cells and can be multiplied indefinitely.As long as they can differentiate into cells of target tissues such as cartilage, bone, muscle, nerve or brain tissue, either can be used in the present invention.Stem cells or progenitor cells useful in the present invention include, but are not limited to, placenta-derived progenitor cells, mesenchymal stem cells, periosteal stem cells, synovium-derived progenitor cells, meniscus-derived progenitor cells, etc.

[0039] "Mesenchymal cells" or "mesenchymal stem cells" ("MSCs") are stem cells that can differentiate into a variety of cell types, such as osteoblasts, chondrocytes, muscle cells, and adipocytes. In some embodiments, MSCs are derived from bone marrow. In some embodiments, MSCs are derived from adipose tissue.

[0040] "Placenta-derived progenitor cells" or "PLC" are progenitor cells derived from the placenta after birth and can be collected by methods known in the art. See, for example, US2008 / 0131410 and US2014 / 0349391 to Hariri and WO2005 / 038012A3 to Ethicon Incorporated, which are incorporated herein by reference. In some embodiments, the use of placenta-derived progenitor cells is preferred due to the favorable characteristics of immune system tolerance, availability, and expansion capacity. As disclosed herein, placenta-derived progenitor cells have been shown to be effective in the present invention, even though previous studies have reported that these cells have shown limited potential for chondrogenic differentiation compared to more obvious cell sources such as bone marrow-derived MSCs. See, for example, Jeon et al., “Comparative Analysis of Human Mesenchymal Stem Cells Derived From Bone Marrow, Placenta, and Adipose Tissue as Sourced of Cell Therapy,” J. Cell Biol. 117(5):1112-1125 (2016).

[0041] "Peripheral blood mononuclear cells" ("PBMCs" or "PBMNCs") are white blood cells with round nuclei and contain a mixture of immune cells such as lymphocytes (T cells, B cells, NK cells), dendritic cells and monocytes. PBMCs can be obtained by methods known in the art, such as by density gradient centrifugation of whole blood or blood apheresis, or buffy coats (white blood cell concentrates).

[0042] PBMCs can be "activated" by incubation with tissue-specific antigens to activate immune cells against that tissue. In some embodiments, PBMCs can be activated by incubating the PBMCs in an activation medium containing tissue antigens for about 1, 2, 3, or 4 days. In some embodiments, the tissue antigen is an enzymatic hydrolysate of the desired tissue (e.g., bone, cartilage, muscle, brain, or nerve tissue). In some embodiments, the activation medium further comprises one or more additives, such as, for example, sugars such as glucose, histamine type 2 receptor blockers (H2RA or H2 antagonists, e.g., famotidine), COX1 / 2 inhibitors (e.g., indomethacin), antibiotics, etc.

[0043] Activated PBMCs (also referred to herein as activated MCs, or effector cells (ECs)) may be identified by detection of stimulation of macrophages and lymphocytes, for example by detection of increased expression of the markers CD68 and CD25, and in some embodiments CD69, and / or increased expression of regeneration-inducing markers and / or associated factors that induce differentiation into the tissue of interest (e.g., COMP and BMP2 for cartilage differentiation).

[0044] Cellular Compositions and Methods of Use Compositions are provided that include activated PBMCs and stem / progenitor cells in co-culture. The PBMCs are activated with antigens corresponding to the tissue to be regenerated ("tissue of interest"), and during the in vitro co-culture of the activated PBMCs and stem / progenitor cells, the stem / progenitor cells are influenced to initiate a pathway of differentiation into a specific tissue. This influence during co-culture results in unexpectedly excellent results of tissue regeneration, with functional recovery upon administration of the co-culture to the tissue, and in some embodiments, rapid (e.g., less than one week) tissue regeneration and functional recovery.

[0045] To provide adequate cellular crosstalk, activated PBMCs and stem / progenitor cells, such as placenta-derived progenitor cells, are provided in the composition at a ratio of 6:1, 5:1, 4.5:1, or 4:1 to 3:1, 2.5:1, or 2:1 PBMC:stem / progenitor cells. In some embodiments, the ratio is 4.5:1 to 2:1. In some embodiments, the ratio is about 4:1. In some embodiments, the ratio is about 3:1. Having activated PBMCs is beneficial in providing synergistic effects in cellular crosstalk, but too high a ratio of activated PBMCs may result in significant apoptosis.

[0046] Administration of cells may be by direct administration to tissue, for example by injection of a drip, or may be systemic administration, where cells (PBMCs and / or stem or progenitor cells) migrate to the site of injury or disease. For example, in the case of cartilage regeneration, cell co-cultures may be injected or injected into the site of injury, such as an osteoarthritic knee, whereby administration (e.g., intra-articular injection) results in functional regeneration of articular cartilage in the knee within a time period of less than one week, less than two weeks, less than one month, less than two months, or less than three months in some embodiments.

[0047] In some embodiments, the co-culture composition also includes a hydrogel carrier (including, for example, collagen, gelatin, fibrin, combinations thereof, etc.) and / or hyaluronic acid.

[0048] Co-cultures may also be used in tissue engineering approaches, such as by seeding cells onto a tissue scaffold, such as a three-dimensional matrix or hydrogel, with the co-culture activated to the tissue of interest to be engineered. In some embodiments, the activated PBMCs and stem / progenitor cells may be seeded onto the scaffold together after co-culture, while in other embodiments, the stem / progenitor cells may be seeded first, followed by the activated PBMCs.

[0049] A "scaffold" on which cells may be seeded and grown to give rise to cultured tissue includes any suitable support and / or three-dimensional matrix. See, for example, U.S. Patent Nos. 6,998,418, 6,485,723, 6,206,931, 6,051,750, and 5,573,784. Preferably, the scaffold is configured to support cell attachment, growth, and / or differentiation thereon. The scaffold may be formed from any suitable material, including, but not limited to, synthetic or natural polymers, other biopolymers, and combinations thereof. In some embodiments, the scaffold comprises a collagen support or an acellularized tissue support. In some embodiments, the scaffold may comprise an electrospun matrix. In some embodiments, the scaffold comprises a polymeric matrix (e.g., collagen, hydrogel, etc.). In some embodiments, the scaffold comprises fibrin or fibrinogen.

[0050] The invention is described in more detail in the following non-limiting examples. EXAMPLES

[0051] Example 1: Functional regeneration of severely damaged osteochondral units. The inventors have developed an immunomodulatory cell treatment that can be injected intra-articularly (IA) early in the onset of PTOA or during disease progression, and has shown significant promise as a treatment in small animal models of PTOA. Our breakthrough treatment is based on a combination of cell products that have shown efficacy when given IA to experimentally injured rats.

[0052] Knee injury results in the immediate activation of proinflammatory neutrophils, which decontaminate and clear the site, leading to the infiltration of mononuclear cells. Cytokines secreted at the wound site drive the differentiation of mononuclear cells into macrophages, which remove apoptotic cells and act as antigen-presenting cells specific to the damaged tissue. This step prevents further leukocyte influx and drives the injury environment toward the regeneration-inducing process. In functional regeneration, the completion of the proinflammatory phase leads to the recruitment of regeneration-inducing immune cells, which then lead to the recruitment of progenitor cells, a critical step for functional healing. Signaling cascades that drive the balance, polarization and subsequent action of recruited immune cells, including helper T (Th)1, Th2 and Th17 cells, as well as regulatory T (Treg) cells, macrophages and mast cells, are important for functional conversion. Thus, we hypothesized that failure of cartilage regeneration may be caused by an imbalance between proinflammatory and regeneration-inducing cells, leading to prolonged inflammation and fibrosis.

[0053] In an effort to study and discover interventions in PTOA, we developed four rat models of osteochondral defect (OCD) and subsequent healing. These four separate models represent different levels of damage and regeneration. To this end, small diameter (SD) and large diameter (LD) full-thickness OCDs were created in the patellofemoral groove of 10-week-old wild-type (WT) and mature T cell-deficient (T cell-) female rats and followed for 12 weeks. Tissue healing, progenitor cell activation and extracellular matrix (ECM) generation were assessed 96 hours after defect creation, as well as 1, 4 and 12 weeks after defect creation. Functional healing was confirmed in SD-WT animals, whereas different degrees of impaired healing and fibrosis were observed in WT-LD, T cell-SD and -LD joints. These experiments demonstrated a direct correlation between the inflammatory response and progenitor cell activation 1 week after injury within the healing status of each model.

[0054] Based on these findings, a cell-based treatment was developed with the aim of facilitating the regeneration induction process. For comparison, three specific cell populations were developed: (1) rat mononuclear cells isolated and activated with cartilage antigens and induced into a chondroactivating effector cell (EC) population, (2) rat placenta-derived progenitor cells (PLC) expanded in vitro, and (3) a combination of EC and PLC in a 4:1 ratio cocultured for 24 h.

[0055] ECs can be obtained from peripheral blood mononuclear cells (PBMNCs, or MNCs) extracted from whole blood or apheresis of patients or donors. PBMNCs are obtained by purification through a Ficoll-Hypaque gradient. PBMNCs were activated in DMEM containing 4500.0 mg / ml glucose and GlutaMAx with 10% hydrolyzed antigen (cartilage), 10 μl / ml famotidine, and 4.5 μl / ml indomethacin for a period of 72 hours. PLCs were obtained from the Clinical Manufacturing Center at the Wake Forest Institute for Regenerative Medicine and were expanded and cultured in growth medium. Co-cultured cell products are obtained through 24-hour coincubation of ECs and PLCs at a 4:1 ratio in serum and antigen-free medium.

[0056] In vitro mechanistic evaluation of chondroactivatable EC confirmed the stimulation of macrophages and lymphocytes as indicated by increased CD68, CD69 and CD25 (Figure 1) compared to primary PBMNC populations. These findings were further supported by gene expression, where increased expression of regeneration-inducing markers TNFalpha and IL10 was observed along with increased expression of chondrogenic differentiation-inducing factors, COMP and BMP2 (Figure 2).

[0057] Upon 24-hour coculture of ECs with PLCs, the initiation of chondrogenic differentiation was confirmed by the transient upregulation of the transcription factor, cartilage matrix protein and synovial lubricant Prg4 (Figure 3). When these cell populations were injected into three models of functional injury healing and then pathologically evaluated 12 weeks after injury creation, the cocultured cell treatments induced functional regeneration, whereas the other cell treatments with each of the cell populations alone did not (Figure 4). This was confirmed by (1) the culminating point between the subchondral bone and the articular unit, (2) glycosaminoglycan-rich subchondral articular cartilage, and (3) the restoration of an articular chondrocyte layer secreting lubricin, a qualitative marker of functional articular cartilage. Administration of placental cells alone produced an anti-inflammatory effect and partial regeneration, but did not induce the rapid tissue repair and functional regeneration as did the cocultured composition (Figure 5).

[0058] These results demonstrate for the first time that functional regeneration of severely damaged osteochondral units, a field-wide goal for 40 years, can be achieved with an injectable immunomodulatory cell-based treatment.

[0059] Example 2: Repeated injections of cell-based treatments CT scans were performed on rat synovial joints after: (A) repeated injections (weeks 1 and 3) of co-cultured chondroactivating mononuclear cells (ECs) and placenta-derived progenitor cells (PLCs), (B) with delayed injection 2 weeks after OCD creation, and (C) with delayed treatment 4 weeks after OCD creation. The results showed that repeated treatments did not induce proliferation of subchondral bone or osteophytes, but delayed treatment at 2 weeks showed satisfactory regeneration of mineralized tissue, and injections at 4 weeks left some defects.

[0060] These data suggest that approximately one week after injury may be the most favorable time for administration, although later time points also show significant improvements in healing.

[0061] Example 3: Nanostring analysis of six donor populations Mononuclear cells from six different patients were activated as chondroactivating effector cells and subjected to nanostring analysis to assess activation towards specific T cell populations. The resulting heatmap confirmed that MNCs from different donors responded very similarly, confirming the expected limited donor variability in co-cultures. Nanostring analysis further confirmed that activation of the six donor populations led to a decrease in the expression of the pro-inflammatory cytokines IL17A, IL17C and IL17F.

[0062] Nanostring analysis further confirmed that the activation of the six donor populations and the subsequent induction of chondrogenic differentiation were associated with activated BMP signaling, as indicated by elevated SMAD2 and SMAD3 expression. SQSTM is a gene involved in tissue remodeling. Specifically, it is an autophagosomal cargo protein that targets other proteins it binds to for selective autophagy by interacting with GATA4 and targeting it for degradation, which may inhibit GATA-4-associated senescence and cellular senescence-associated secretory phenotypes, and is also involved in the recycling of aged cell parts and unnecessary proteins, the self-destruction of cells (apoptosis), and the body's immune and inflammatory responses. Without wishing to be bound by theory, based on this upregulation, this may be an important factor underlying the potential regeneration-inducing effect seen in ECs.

[0063] Example 4: Compassionate Use Trials in Human Patients Nine patients (6 males, 3 females) were enrolled in a compassionate use trial and received injections of a cell-based treatment using autologous adipose-derived cells as stem / progenitor cells. Patients offered treatment were at least 18 years old, presented to the clinic for knee osteoarthritis (KOA), weighed more than 45 kg, and had blood parameters of hematocrit ≥ 35%, MCV ≥ 70%, MCH ≥ 31%, and leukocytes ≥ 4000 / mm3. 3 , Platelets: 150000~400000 / mm 3and had normal cardiac, hepatic, respiratory, and renal function.Inclusion criteria: definite diagnosis of KOA (effusion and synovial thickening / synovitis, subchondral bone marrow edema and / or cysts, loss of cartilage quality (partial or full thickness) bursitis, iliotibial band syndrome) supported by clinical symptoms and knee abnormalities on x-ray and confirmed by MRI of the knee. MRI features and associated pain were >80% according to the KOOS scale.Exclusion criteria: active neoplasm, active systemic infection viral, bacterial or fungal (internal) at the start of treatment, HIV, Hepatitis B or C positive, patients with jaundice or liver failure, pregnant patients, or individuals with alcohol or drug dependency.

[0064] All included patients suffered from severe KOA. Total knee arthroplasty was indicated because OA progression did not result in treatment response to standard palliative therapy. Treatment was performed under a compassionate use trial. Patient safety and clinical follow-up were monitored at least once a week to observe the safety, efficacy, and effectiveness of the treatment. Follow-up controls were performed on days 60, 90, and 180, and data from day 180 were used in this study. Safety was evaluated according to the National Cancer Institute. Common Terminology Criteria for Adverse Events (version 5.0), KOOS index, was assessed at 3 months. MRI of the treated area was performed 6 months after the end of treatment. Statistical analysis of clinical data was performed with MedCalc software from MedCalc Inc.

[0065] As shown in FIG. 6, 180 days after treatment, there was a significant improvement in pain, functionality and quality of life reported by the patients.

[0066] The foregoing is illustrative of the present invention and is not to be construed as limiting thereof, The present invention is defined by the following claims and equivalents of the claims included therein.

Claims

1. a) activated peripheral blood mononuclear cells (PBMCs), and b) Stem or progenitor cells A composition comprising a co-culture of The composition, wherein the activated PBMCs and the stem or progenitor cells are present in the composition at a ratio of activated PBMCs:stem or progenitor cells of from 6:1, 5:1, 4.5:1 or 4:1 to 3:1, 2.5:1 or 2:

1.

2. The composition of claim 1 , wherein the stem or progenitor cells are placenta-derived progenitor cells.

3. The composition of claim 1 , wherein the stem or progenitor cells are mesenchymal stem cells.

4. The composition of claim 1 , wherein the activated PBMCs are from a different subject than the stem or progenitor cells.

5. The composition of claim 1 , wherein the activated PBMCs and the stem or progenitor cells are both derived from the same subject.

6. The composition of claim 1, wherein the activated PBMCs are activated with an antigen derived from a tissue selected from the group consisting of cartilage, bone, muscle, nerve, and brain tissue.

7. The composition of claim 1 , wherein the activated PBMCs are activated with an antigen derived from a tissue selected from the group consisting of cartilage and bone tissue.

8. The composition of claim 6 , wherein the antigen is an enzymatic hydrolysate of the tissue.

9. 10. The composition of claim 1, wherein the composition is formulated for injection or infusion into damaged or diseased tissue.

10. The composition of claim 1 , wherein the activated PBMCs and the stem or progenitor cells are co-cultured for less than 96, 72, or 48 hours (e.g., about 24 hours) prior to use.

11. The composition of claim 1 , further comprising a hydrogel carrier and / or hyaluronic acid.

12. A composition described in any one of claims 1 to 11 for regenerating tissue in a subject in need of tissue regeneration.

13. 13. The composition of claim 12, wherein the activated PBMCs are obtained by extraction from the subject's or donor's whole blood, apheresis, or buffy coat.

14. 13. The composition of claim 12, wherein the PBMCs are autologous with respect to the subject in need of regeneration of the tissue, and the stem or progenitor cells are allogeneic with respect to the subject in need of regeneration of the tissue.

15. The composition of claim 12, wherein the composition is administered locally to the site of injury or disease in a subject in need of regeneration of the tissue.

16. 16. The composition of claim 15, wherein the site of injury or disease is the subject's knee.

17. 13. The composition of claim 12, wherein the composition is administered systemically and the PBMCs and / or stem or progenitor cells migrate to the site of the injury or disease in a subject in need of tissue regeneration.

18. 18. The composition of claim 17, wherein the site of injury or disease is the subject's knee.

19. 1. A method for forming tissue in vitro, comprising: providing a tissue scaffold; and seeding the scaffold with stem or progenitor cells and activated PBMCs, whereby the stem or progenitor cells differentiate into tissue in vitro.

20. 20. The method of claim 19, wherein the activated PBMCs are activated with an antigen derived from a tissue selected from the group consisting of cartilage, bone, muscle, nerve and brain tissue.

21. 21. The method of claim 19 or claim 20, wherein the stem or progenitor cells and activated PBMCs are seeded together onto the tissue scaffold as a co-culture.

22. 21. The method of claim 19 or claim 20, wherein the stem or progenitor cells and activated PBMCs are seeded separately onto the tissue scaffold.

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