Cells and methods

Abstract

The present invention relates to a population of cells, said population of cells comprising at least 50% macrophage or monocyte cells, characterized in that at least 50% of said macrophage or monocyte cells express each of the markers: MRC1; TIE2; and CD163. The present invention also relates to uses of these cells, methods of making them, and methods of treating a subject by administering them.

Description

[Background technology]

[0001] Peripheral arterial disease affects 20% of individuals over the age of 75. It can lead to critical limb ischaemia (CLI), which manifests as intractable pain, ulcers and gangrene. The quality of life of patients with CLI is comparable to that of patients with terminal cancer. Each year, 40,000-60,000 patients are diagnosed with CLI in the UK. One third of these patients cannot be revascularized by conventional treatments such as bypass surgery / stent placement and require amputation. The estimated total number of CLI patients worldwide is approximately 237 million.

[0002] Therapeutic cell-based neovascularization has been highlighted as a promising treatment for the salvage of critically ischemic limbs, aiming to stimulate the growth of new blood vessels in ischemic tissues. However, numerous clinical trials of cell therapy have reported only modest efficacy so far, because poorly defined and heterogeneous populations of cells (mononuclear cells) from bone marrow or peripheral blood have been used with little understanding of their activity (Fadini et al. "Autologous stem cell therapy for peripheral arterial disease meta-analysis and systematic review of the literature." Atherosclerosis 2010 March 209(1);Qadura et al. 2018 Stem Cells Feb 36(2) "Cell Therapy for Critical Limb Ischemia: An Integrated Review of Preclinical and Clinical Studies.").

[0003] It is widely accepted that effective therapy will likely require more specific and potent cell types that can be reliably isolated in sufficient numbers, which is a problem in the art.

[0004] A small and specific subpopulation of mononuclear cells, monocytes / macrophages (Mo / MΦ), has been investigated, and it has been shown that those expressing the marker TIE2 may regulate revascularization after ischemia (Patel et al. 2013 "TIE2-expressing monocytes / macrophages regulate revascularization of the ischemic limb." EMBO Mol Med 2013 May 7). Unfortunately, the number of these cells that can be isolated from blood (approximately 3% of the total monocyte population) is too small to provide effective therapy.

[0005] Rybalko et al. 2017 (Regen. Med. Volume 12, number 2, pages 153-167) describes the therapeutic potential of adipose tissue derived stem cells and macrophages for ischemic skeletal muscle repair. Rybalko et al. use a transwell approach in engineering their cells. Rybalko et al. take more than 14 days to produce their cells. There is no disclosure of triple positive MRC1 / TIE2 / CD163 positive cells in Rybalko et al.

[0006] Kim and Hematti 2009 ("Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages." Exp Hematol December;37(12):1445-53 (corresponding to published US patent application US2011 / 0045071 by Hematti and Kim)) disclose a method that involves first converting primary monocytes into macrophages by culturing for 7 days without cytokines (see page 1446 - left column - last paragraph under "Cell Culture") and then culturing those macrophages with MSCs. Kim and Hematti. took more than 10 days to produce their cells. There is no disclosure of triple positive MRC1 / TIE2 / CD163 positive cells in Kim and Hematti. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] U.S. Patent Application No. US2011 / 0045071 [Non-patent literature]

[0008] [Non-Patent Document 1] Fadini et al. "Autologous stem cell therapy for peripheral arterial disease meta-analysis and systematic review of the literature." Atherosclerosis 2010 March 209(1) [Non-Patent Document 2] Qadura et al. 2018 Stem Cells Feb 36(2) "Cell Therapy for Critical Limb Ischemia: An Integrated Review of Preclinical and Clinical Studies." [Non-Patent Document 3] Patel et al. 2013 "TIE2-expressing monocytes / macrophages regulate revascularization of the ischemic limb." EMBO Mol Med 2013 May 7 [Non-Patent Document 4] Rybalko et al. 2017(Regen. Med. Volume 12, number 2, pages 153-167) Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention seeks to overcome the problems associated with the prior art. [Means for solving the problem]

[0010] The present invention relates to cells that have been "educated" or "primed" or otherwise driven to express the key markers MRC1, TIE2 and CD163. The cells of the invention exhibit advantageous functional properties, for example in revascularization. Thus, the cells of the invention have direct industrial application, for example in clinical settings where revascularization is required in peripheral vascular diseases, such as critical limb ischemia (CLI). Most appropriately, "educating" or "priming" refers to the co-culture of starting cells (monocytes / macrophages) with mesenchymal stem / stromal cells (MSCs), more appropriately mesenchymal stem cells. Suitably, the cells are human cells. Suitably, the cells are human cells, in vitro or ex vivo. Suitably, the cells are not part of a human body. Suitably, the cells are not germline cells. Suitably, the cells are not obtained by an essentially biological process, but rather with in vitro manipulation. Suitably the cell is not derived from a human embryo, suitably not derived from a human embryo.Suitably the cell is not a totipotent cell.

[0011] Briefly, such new cells are produced by starting with subject-derived monocytes / macrophages and manipulating them in vitro using a carefully designed method. Critical steps in the method of the invention may include the ratio of monocytes / macrophages to stem cells in the co-culture step, and / or the length of time of the culture step, and / or the number of medium changes used during this culture period. The inventors have designed the method of the invention thoughtfully based on well-founded research and intellectual deliberation. The inventors have discovered a surprising optimal window in the process where it is possible to stimulate marker expression (and therefore cell function). The particularly surprising nature of this development is demonstrated by the fact that continued culture over a longer period leads to the loss of said markers. Thus, the present invention allows the production of cells with new phenotypic characteristics and therapeutically beneficial functions, making a new and surprising contribution to the art.

[0012] The present invention is based on these surprising advances.

[0013] Thus, in one embodiment, the invention provides a population of cells, the population of cells comprises at least 50% myeloid cells, e.g., macrophages or monocytes; At least 50% of the bone marrow cells, e.g., macrophages or monocytes, express the marker: · MRC1; TIE2; and CD163 The present invention is characterized in that it expresses each of the above.

[0014] Suitably, the population of cells comprises at least 70% myeloid cells, such as macrophages or monocyte cells.

[0015] Suitably, the population of cells comprises at least 80% myeloid cells, such as macrophages or monocyte cells.

[0016] In another embodiment, the present invention relates to a population of cells as described above, At least 60% of the bone marrow cells, e.g., macrophages or monocytes, express the marker: · MRC1; TIE2; and CD163 Each of the above is expressed.

[0017] In another embodiment, the present invention relates to a population of cells as described above, wherein said macrophages or monocyte cells express CD14 and / or CD45.

[0018] In another aspect, the invention provides a population of cells, The population of cells comprises at least 50% CD14+ and / or CD45+ cells, and at least 50% of the CD14+ and / or CD45+ cells express the marker: · MRC1; TIE2; and CD163 The present invention is characterized in that it expresses each of the above.

[0019] Suitably, the population of cells comprises at least 70% CD14+ and / or CD45+ cells. Suitably, the population of cells comprises at least 80% CD14+ and / or CD45+ cells.

[0020] In another embodiment, the present invention relates to a population of cells as described above, At least 60% of said CD14+ and / or CD45+ cells have the marker: · MRC1; TIE2; and CD163 Each of the above is expressed.

[0021] In another embodiment, the invention relates to a population of cells as described above, wherein said CD14+ and / or CD45+ cells are or are derived from bone marrow cells.In another embodiment, the invention relates to a population of cells as described above, wherein said CD14+ and / or CD45+ cells are or are derived from circulating leukocytes.

[0022] In one embodiment, suitably the cell is an in vitro cell or an ex vivo cell.

[0023] Suitably, the population of cells described above comprises a 3:1 ratio of monocyte / macrophage cells (or CD14+ and / or CD45+ cells):MSCs.

[0024] Suitably, the population of cells described above comprises a 3:1 ratio of triple positive monocyte / macrophage cells (or triple positive CD14+ and / or CD45+ cells):MSCs.

[0025] Suitably, the population of cells described above comprises macrophages or monocytes that express TNF alpha (TNFa) and / or IL-12. Suitably, at least 60%, more suitably at least 80%, more suitably at least 90% of said CD14+ and / or CD45+ cells further express IL-12. Suitably, at least 4%, more suitably at least 5%, more suitably more than 5% of said CD14+ and / or CD45+ cells further express TNF alpha (TNFa).

[0026] Suitably, the population of cells described above comprises cells which are encapsulated.

[0027] In another embodiment, the present invention provides (a) providing macrophages or monocytes (or CD14+ and / or CD45+ cells) from a subject (b) providing the MSC; (c) culturing said macrophages or monocytes (or CD14+ and / or CD45+ cells) with said MSCs The present invention relates to a method comprising the steps of:

[0028] Suitably the method is a method of inducing expression of MRC1, TIE2 and CD163 in macrophages or monocytes (or CD14+ and / or CD45+ cells).

[0029] Suitably the method is a method for producing triple positive macrophages or monocytes (or triple positive CD14+ and / or CD45+ cells).

[0030] Suitably, step (c) comprises contacting said macrophages or monocytes (or CD14+ and / or CD45+ cells) with said MSCs to produce a cell mixture and culturing said cell mixture.

[0031] Suitably therefore the present invention comprises: (a) providing monocytes from a subject; (b) providing the MSC; (c) culturing said monocytes with said MSCs. Regarding the method, Step (c) comprises contacting said monocytes with said MSCs to produce a cell mixture and culturing said cell mixture.

[0032] Suitably, the ratio of (macrophages or monocytes) or (CD14+ and / or CD45+ cells):(MSCs) in step (c) is 3:1.

[0033] Suitably, the cells are cultured for about 3 to 7 days.

[0034] Suitably, the cells are cultured for about 3 to 5 days.

[0035] Suitably the cells are cultured for about 3 days.

[0036] The cells are suitably cultured for an interval that effectively induces triple positive marker expression.

[0037] In one embodiment, the cells may be cultured for about 3 to 7 days.

[0038] In a more suitable embodiment, the cells may be cultured for about 3-5 days, which has the advantage of producing more triple positive cells than a 7 day culture.

[0039] In the most suitable embodiment, the cells may be cultured for about 3 days, which has the advantage of producing the greatest proportion of triple positive cells.

[0040] In one embodiment, the cells may be cultured for about 72 to 168 hours.

[0041] In a more suitable embodiment, the cells may be cultured for about 72 to 120 hours, which has the advantage of producing more triple positive cells than a 168 hour culture.

[0042] In the most suitable embodiment, the cells may be cultured for about 3 days (about 72 hours), which has the advantage of producing the greatest proportion of triple positive cells.

[0043] Suitably, the cells are cultured in medium, which may be changed every 3 to 5 days.

[0044] In one embodiment, suitably the medium is changed every 3 days.

[0045] In one embodiment, suitably the medium is changed every 4 days.

[0046] In a particularly suitable embodiment, suitably the medium is changed every 5 days.

[0047] In one embodiment, suitably the medium is changed every 7 days.

[0048] Most suitably, if the cells are cultured for more than 5 days, the medium is changed no later than the 5th day.

[0049] An advantage of the present invention is that induction of triple marker expression is efficient, such that little cultivation of the cells beyond day 5 is required - and therefore, advantageously, medium changes are correspondingly less required, thereby saving costs and effort and avoiding the risk of contamination during medium changes.

[0050] According to the method of the present invention, maximum / optimal triple positive marker expression is obtained on day 3 of culture. A further advantage of the present invention is that this optimal triple positive marker expression is maintained on day 4 and maintained on day 5.

[0051] Also successfully observed is that triple positive marker expression can continue into the sixth day and in some cases into the seventh day.

[0052] Of note, triple positive marker expression declines from day 7 onwards, so cells cultured to day 7 are useful, although perhaps higher doses of such cells could be delivered to patients to maintain efficacy.

[0053] Suitably the cells are not cultured for 8 days or any further days. Suitably the cells are not used if they have been cultured for 8 days or any further days.

[0054] Suitably the cells are cultured for at least day 3. Suitably the cells are not used until they have been cultured for at least day 3.

[0055] The present invention also relates to a population of cells obtainable by the method described above.

[0056] The present invention also relates to a population of cells obtained by the method described above.

[0057] In another embodiment, the present invention relates to a population of cells as described above for use in the treatment of peripheral vascular disease, more suitably critical limb ischemia (CLI), more suitably global severe chronic limb ischemia (CLTI, chronic limb threatening ischaemia).

[0058] In another embodiment, the present invention relates to a population of cells as described above for use in the treatment of fibrosis.

[0059] In another embodiment, the invention relates to a population of cells as described above for use in the treatment of ischemic stroke.

[0060] Suitably, the treatment comprises administering to the subject a population of cells. Suitably, the treatment comprises administering to the subject about 10 6 ~10 9 Suitably, the treatment comprises administering a dose of said cells by injection.

[0061] In another embodiment, the present invention relates to a method of treating a mammalian subject comprising administering to said subject a population of cells as described above.

[0062] Suitably, the method comprises the steps of: 6 ~10 9 The method includes administering a dose of said cells.

[0063] In another embodiment, the invention relates to the use of the population of cells described above for inducing angiogenesis in a mammal.

[0064] Also described are compositions comprising a population of cells described above.

[0065] Suitably, the composition is a pharmaceutical composition.

[0066] Also described is the use of the above described compositions in medicine. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0067] One advancement the inventors have made is the creation of an ex vivo method to generate large numbers of specific populations of monocytes / macrophages (Mo / MΦ) expressing the marker TIE2 capable of stimulating new blood vessel growth for delivery to the ischemic limb.

[0068] To do this, we exploit the immunomodulatory effects of mesenchymal stem / stromal cells (MSCs). MSCs can be obtained from either bone marrow (BM) or adipose tissue. MSCs modulate the properties of cells in their vicinity, including Mo / MΦs, by shifting their polarization from a pro-inflammatory to an anti-inflammatory phenotype (Kim and Hematti 2009 "Mesenchymal stem cell-educated macrophages: a novel type of alternatively activated macrophages." Exp Hematol December;37(12):1445-53 (corresponding to published U.S. Patent Application No. US2011 / 0045071 by Hematti and Kim); Maggini et al. 2010 "Mouse bone marrow-derived mesenchymal stromal cells turn activated macrophages into a regulatory-like profile." PLoS One 5(2):e9252.).

[0069] In Kim and Hematti 2009, they first transform primary monocytes into macrophages by culturing for 7 days without cytokines (see page 1446 - left column - last paragraph under "Cell Culture"). Only then are the resulting macrophages exposed to MSCs. In contrast, in the method of the present invention, primary monocytes are directly cultured / contacted with MSCs on day 0. Thus, according to the present invention, bone marrow cells, e.g. primary monocytes, are placed in culture immediately after isolation - the start time is the time when bone marrow cells are placed in culture. In the present invention, primary monocytes are placed in culture with MSCs, so that at this point there are no pre-differentiated macrophages in the mixture. In the prior art method, first macrophages are generated and then (subsequently) these macrophages are contacted with MSCs. In one method distinct from the present invention herein, Kim and Hematti 2009 first place their monocytes in culture for 7 days to differentiate the monocytes into macrophages, and then it is these macrophage cells (i.e., pre-differentiated macrophages) that are exposed to MSCs.

[0070] We believe that the cells of Kim & Hematti 2009 are not the cells described herein, i.e., they are not triple expressors of MRC1 (CD206), TIES2 (CD202B) and CD163. The reason for this is, firstly, that the cells of the present invention show differences in the expression markers. This is because only a small percentage of the cells of Kim & Hematti express even one of the markers (CD206), which is typical for low CD206 expressors. For example, there is a wide range of CD206 expression in Kim & Hematti (Figure 3 - A / C / E) from 11.81% to 75.89%, which indicates that CD206 expression is unreliable and that the average CD206 expression is low. (The average amount is 46.1%). In contrast, our data show that our cells are consistent (reliable) high CD206 expressers with an average CD206 expression of 92% (+ / - SEM 14%) (Re: 92% - we see Figure 2B and Figure 11B). We also show that our method is reproducible across different technicians (experienced technicians) and still results in expression of over 90% (see Figure 4).

[0071] The advantage of the present invention is that the monocytes / macrophages described herein expressing CD206 have a tissue remodeling phenotype and play an important role in the formation of new blood vessels (both angiogenesis and arteriogenesis). The cells of the present invention also have important anti-fibrotic functions. This is demonstrated in the Examples section.

[0072] Second, the cells of the present invention exhibit different properties. For example, the cells described herein exhibit high TNFa (TNF alpha) levels, whereas Kim and Hematti 2009 (US2011 / 0045071) have low TNFa (TNF alpha) levels. TNFa is important in tissue remodeling. TNFa has a role in pathological angiogenesis and arteriogenesis. TNFa stimulates the growth of collateral vessels in post-ischemic tissues. TNFa has also been shown to have significant anti-fibrotic functions, regulating the resolution phase of fibrosis.

[0073] Kim and Hematti's cells downregulated TNFα; Kim and Hematti disclose that the cells are downregulated by one-third compared to control cells. Indeed, their data (Kim and Hematti, Figs. 3E and 3F) show that only 3% of their cells express TNFα. The levels of TNFα-expressing cells fall below control cells, whereas the cells of the present invention show constant or elevated levels of TNFα expression, and thus the cells of the present invention do not apparently downregulate TNFα as in Kim and Hematti.

[0074] We quantified expression levels: control cells express a median of 42 pg / mL, whereas our MSC-primed cells (cells of the invention) express high levels - a median of 251 pg / mL - thus showing that our cells of the invention upregulate TNFα levels by 6-fold.

[0075] IL-12 is another potent cytokine that induces tissue remodeling. Kim and Hematti's cells lose IL-12 expression, while IL-12 levels are still high in our cells. We refer to Figure 27: Day 3: No difference in IL-12 expression (IL-12 is still highly expressed in our cells after co-culture, while Kim & Hematti's cells do not express IL-12); Day 7: No difference in IL-12 expression (IL-12 is still highly expressed in our cells after co-culture, while Kim & Hematti's cells do not express IL-12 (e.g., only approximately 1.5% of Kim and Hematti's cells express IL-12)).

[0076] The difference in these two cytokines alone indicates that the cells of the present invention have properties that are quite different from the cells of the prior art such as Kim & Hematti, etc. This is especially true because TNFα and IL-12 are pro-inflammatory cytokines, the expression of which would not be expected.

[0077] Please note that unless otherwise clear from the context, the terms high and low used to describe expression refer not to the absolute level of expression but to the percentage of expressing cells - e.g., the percentage of cells deemed to be expressing by FACS analysis or similar binary counting methods. This will be clear to the skilled artisan, since polypeptides such as TNFα / IL-12 are secreted and therefore assessing the level of expression can be a challenge to compare between different cells, e.g., if cells are grown in a large volume of medium, then the apparent concentration of expressed protein per cell will be measured to be lower (and vice versa), and therefore expression levels are most often assessed as the percentage of expressing cells unless otherwise stated.

[0078] We disclose techniques for culturing monocytes with MSCs in order to prime ("educate") monocytes, i.e., to switch monocytes towards a distinct pro-angiogenic / pro-arteriogenic monocyte / macrophage (Mo / MΦ) phenotype.

[0079] In one embodiment, "primed" or "educated" monocytes / macrophages refers to monocytes / macrophages that have been co-cultured with MSCs, suitably monocytes / macrophages that have been co-cultured with MSCs in vitro.

[0080] In addition, the present inventors have MRC1, and TIE2, and CD163 It is hereby disclosed for the first time that "triple positive" monocytes / macrophages (Mo / MΦ) expressing each of the above are extremely clinically useful as described herein.

[0081] Disclosed are conditions that allow the highest percentage of triple positive cells to be obtained from the starting cells.

[0082] What is demonstrated is that triple positive macrophages can be used to stimulate the production of new blood vessels (revascularization).

[0083] We disclose a method to produce triple expression of three key markers on macrophages.

[0084] Disclosed is a method for generating triple-expressing macrophages from mononuclear cells taken from a patient's blood.

[0085] The inventors demonstrate a clear link of cells expressing the three markers disclosed herein to the functional advantage of stimulating new blood vessels.

[0086] The cells described herein are novel by virtue of expressing a triple marker combination.

[0087] The sequences of the key markers are publicly available and available to the skilled artisan, as shown in the table below: [Table 1] JPEG2025505710000003.jpg83170

[0088] Expression threshold: Suitably, "expressing" a marker means that the protein can be detected by immunostaining, such as antibody staining, for example flow cytometry.

[0089] Unless otherwise clear, expression refers to expression of a full-length protein or to expression of a biologically significant portion, e.g., a biologically active portion. In one embodiment, expression refers to expression of a marker on the cell surface. In one embodiment, if the marker is an external / cell surface protein, expression refers to expression of the marker sufficient to allow detection on the external cell surface.

[0090] If any further guidance is needed, a normal macrophage or monocyte produces proteins such as CD14 and / or CD45; a cell is considered a monocyte or macrophage if it "expresses" CD14 and / or CD45 as detected by FACS using anti-CD14 and / or anti-CD45 reagents, such as anti-CD14 and / or anti-CD45 antibodies or anti-CD14 and / or anti-CD45 affimers. Cells expressing CD14 or CD45 are designated as CD14+ or CD45+, as is customary in the art. If only one of CD14 / CD45 is used, suitably only CD45 is used, which has the advantage of superior technical performance / ease.

[0091] For markers not normally expressed on macrophages or monocytes, the expression of which is induced by the present invention (e.g., MRC1, TIE2, CD163), cells are said to "express" a marker in accordance with the present invention (i.e., are positive for the marker) if the flow cytometer detects a higher threshold amount of fluorescence than the same cells that do not express the marker (i.e., a negative control). Selecting an appropriate fluorescence threshold is routine for one of skill in the art.

[0092] If any further guidance is needed, a suitable negative control would be a sample of macrophages or monocytes that have not been co-cultured with MSCs (mesenchymal stem cells / stromal cells) as taught herein. For example, the negative control could be a population of unprimed / untreated macrophages or monocytes isolated from peripheral blood.

[0093] More suitably, the negative control may be a population of primary monocytes isolated from peripheral blood. Suitably, the negative control cells are not cultured with MSCs. Suitably, the negative control cells are not cultured in vitro at all (primary monocytes). Monocytes isolated from blood are a very useful negative control, as they do not triple express the markers of the invention.

[0094] Macrophages expressing these three markers in combination have, to the inventors' knowledge, never been described before.

[0095] Suitably, the term "about" applied to a numerical value means + / - 1% of the stated value.

[0096] Suitably, the term mesenchymal stem cell has its natural meaning in the art. The abbreviation "MSC" means mesenchymal stem cell / stromal cell (MSC), more suitably, mesenchymal stem cell.

[0097] Monocytes (Mo) and macrophages (MΦ) Monocytes (Mo) are defined in humans as blood mononuclear cells with a bean-shaped nucleus that express CD11b and CD14 (LPS receptor subunits). Nonclassical monocytes may express lower levels of CD14 plus CD16, and intermediate monocytes express CD14 and CD16. Monocytes (Mo) differentiate into macrophages (MΦ).

[0098] CD14 is considered a unique marker for monocytes (Mo), i.e., if CD14 is found on a circulating cell, it is designated a monocyte (Mo). Monocytes (Mo) also present other markers such as CD45, CD11b (or both).

[0099] Macrophages are a diverse group of white blood cells known to eliminate pathogens through phagocytosis. Human macrophages normally express CD14, CD40, CD11b, CD64, EMR1, lysozyme M, MAC-1 / MAC-3, 25F9, and CD68.

[0100] The cells of the invention also express other markers, including HLA-DR and CD38 (see FIG. 7).

[0101] Traditionally, macrophages have been classified according to the organ in which they are found (e.g., Kupffer cells in the liver, Langerhans cells in the skin, etc.). However, current nomenclature has shifted away from organ-specific naming of macrophages to "M1" and "M2" macrophages. This classification is based on macrophage polarization rather than on their location.

[0102] M1 macrophages are classically activated, usually by IFN-γ or lipopolysaccharide (LPS), to produce proinflammatory cytokines, phagocytose microorganisms, and initiate immune responses. M1 macrophages produce nitric oxide (NO) or reactive oxygen intermediates (ROI) to protect against bacteria and viruses.

[0103] M2 macrophages are selectively activated by exposure to certain cytokines, such as IL-4, IL-10, or IL-13. M2 macrophages will produce either polyamines, which induce proliferation, or proline, which modulates collagen production. These macrophages are associated with wound healing and tissue repair. There are three types of M2 macrophages: M2a, M2b, and M2c. M2 macrophages also contribute to the formation of extracellular matrix and do not produce nitric oxide or present antigens to T cells. Although tumor-infiltrating macrophages are usually classified as M2, some classify them as myeloid-derived suppressor cells (MDSCs).

[0104] There are conflicting opinions in the art as to which subset of macrophages is most effective. In contrast, the inventors' approach is not to generate a specific subset that falls into either the "monocyte" or "M1" or "M2" macrophage categories, but to produce macrophages that express three specific markers (TIE2 and CD163 and MRC1). Thus, in the discussion of the cells of the invention in this document, "monocytes" and "macrophages" are often referred to together (e.g., "Mo / MΦ").

[0105] In strict classification, the circulating leukocytes described herein that are isolated and then co-cultured with MSCs can be seen as pure monocytes since they are circulating, whereas macrophages are usually considered tissue cells. However, the present invention is not concerned with theoretical questions such as (for example) whether the starting cells were differentiated into macrophages during the culture. It can be observed that the starting cells, although starting to differentiate, still have monocytic characteristics (CD14 expression). Indeed, a decrease in the expression of MMP-9 (a matrix metalloprotease that increases as monocytes start to differentiate into macrophages) is seen compared to monocytes cultured alone (Figure 18). Without wishing to be bound by theory, they can be said to be "myeloid" cells, since this encompasses both Mo / MΦ. For the purposes of the present invention, the starting cells (i.e. blood mononuclear cells) can include monocytes or macrophages or a mixture of monocytes and macrophages.

[0106] More suitably, the starting cells (ie blood mononuclear cells) may comprise monocytes, such as primary monocytes.

[0107] Primary monocytes are defined as CD14 expressing monocytes that have been isolated from blood and have not yet been placed in culture (i.e., are still in suspension). As soon as monocytes are placed in a culture dish, they become fixed (adherent) and slowly begin to differentiate into macrophages, so they are no longer primary monocytes. Monocytes incubated in vitro were primary monocytes before they were cultured.

[0108] Suitably, the cells of the invention are co-cultured with MSCs for 100% of their culture time, i.e. the starting cells (monocytes, such as primary monocytes in suspension) are mixed with MSCs, placed into culture on day 0 and co-cultured with MSCs for their entire time in culture. In prior art methods, macrophages are first prepared, usually by culturing monocytes, such as primary monocytes, for 7 days, and only then are the macrophages contacted with MSCs. Suitably, the cells of the invention are co-cultured or have been co-cultured with MSCs for 100% of their culture time. In prior art methods such as Kim & Hematti, they co-culture their macrophages with MSCs for only 3-4 days out of a total of 10-11 days of culture. Thus, at maximum, Kim & Hematti co-culture their macrophages with MSCs for about 30% (3 / 10 days) to 36% (4 / 11 days) of the total culture time.

[0109] Should further guidance be needed, suitably the cells of the present invention are derived from circulating leukocytes.

[0110] Suitably the cells of the invention are bone marrow cells.

[0111] Suitably, the cells of the invention are CD14+.

[0112] Suitably, the cells of the invention are CD45+.

[0113] Most suitably, the cells of the invention are CD14+ and CD45+.

[0114] Most importantly, the cells of the invention are non-naturally occurring "triple positive" cells. Because these cells do not occur in nature, it is not always useful to classify them as "monocytes" or "macrophages," and therefore they are usually referred to collectively as "Mo / MΦ." In one embodiment, the cells of the invention are triple positive monocytes. In one embodiment, the cells of the invention are triple positive macrophages.

[0115] Macrophage "polarization" for tissue repair / remodelling has been mentioned in the prior art. However, the macrophages reported in the prior art express one or more "M2" markers. Suitably, the cells of the invention do not exclusively express M2 markers. Suitably, the cells of the invention are not exclusively M1 macrophages. Suitably, the cells of the invention are not exclusively M2 macrophages. Suitably, the cells of the invention are not exclusively M2a macrophages. Suitably, the cells of the invention are not exclusively M2b macrophages. Suitably, the cells of the invention are not exclusively M2c macrophages.

[0116] Suitably the cells of the invention are or are derived from blood mononuclear cells.

[0117] Suitably, the cells of the invention are human cells.

[0118] Suitably, the cells of the invention are CD45+ (ie suitably the cells of the invention express the CD45 marker).

[0119] Most suitably, the cells of the invention are CD14+ (ie suitably the cells of the invention express the CD14 marker).

[0120] Suitably the cells of the invention are bone marrow cells.

[0121] Suitably, the cell of the invention is a monocyte or a macrophage.

[0122] Suitably the cells of the invention are monocytes.

[0123] Suitably the cell of the invention is a macrophage.

[0124] We teach a method to generate CD45+ Mo / MΦs that express TIE2, MRC1 and CD163 in combination. The cells of the invention can robustly promote blood vessel growth in ischemic tissues. The cells of the invention are very rare in the circulation (less than 1% of monocytes), and therefore the invention has the advantage of allowing the production of up to 1-2 billion such cells in a single cycle / from a single starting pool of Mo / MΦs from leukapheresis by educating the entire monocyte population with MSCs. An additional advantage of this approach is that such cells can be produced and then cryopreserved (e.g. in batches) so that patients can have a long course of autologous therapy with multiple injections over the course of weeks / months. This is not possible with the limited number of CD45+ Mo / MΦs that may occur naturally. The autologous nature of the therapy reduces or eliminates the patient's risk of adverse immune reactions.

[0125] Although the generation of "M2" macrophages has been disclosed, known methods require long-term culture using a combination of growth factors, which is a problem in the art. In contrast, the present invention circumvents the need for growth factors and provides the ability to generate cells of the invention with the specific phenotype and function of the invention within as little as five days. This allows for rapid treatment of patients who often present as emergencies, which are acute exacerbations of chronic diseases.

[0126] Thus, in contrast to prior art approaches such as co-delivery of mouse adipose tissue-derived stem cells and macrophages to stimulate angiogenesis in ischemic mouse limbs, our approach uses MSCs as a way to prime monocytes. MSCs are only used in the process of creating "triple positive" monocytes / macrophages and are not part of the final therapeutic product. In one embodiment, suitably, some residual MSCs may remain in the population of cells administered. More suitably, MSCs are removed such that the cells administered essentially consist of or consist of triple positive macrophage cells and / or triple positive monocyte cells.

[0127] co-culture To the best of the inventors' knowledge, there is no known prior disclosure that the inventive "triple expressing" (TIE2, MRC1 and CD163) monocytes can be generated by co-culture with MSCs. Furthermore, the inventors have also determined the optimal conditions for generating the inventive potent population of cells, which are disclosed herein.

[0128] To the best of the inventors' knowledge, there is no data in the literature regarding how the cells of the invention can be generated from total monocyte populations, or even that their generation is possible.

[0129] The inventors tested a series of four different time points as well as a series of four different ratios of monocytes:MSCs to determine successful conditions for generating the cells of the invention (see Example 5).

[0130] Prior to the present invention, it was similarly unclear whether monocytes from patients with peripheral arterial disease (such as CLI) could even be primed in this manner.

[0131] The teachings and experimental data herein indicate that the immunogenic properties exerted by MSCs are not limited to educating monocytes from healthy adults, and further demonstrate that monocytes from elderly patients with multiple comorbidities can be used to generate the cells / cell populations of the present invention for autologous cell therapy.

[0132] Co-culture conditions Suitably, the isolated monocytes / macrophages and / or the cell mixture of monocytes / macrophages and MSCs are cultured in monocyte medium.

[0133] Exemplary monocyte media include: RPMI-1640 ('Roswell Park Memorial Institute'-1640) supplemented with 10% foetal calf serum (FCS) and optionally supplemented with 1% antibiotic / antimycotic.

[0134] Suitably the cells are cultured in a humidified incubator.

[0135] Suitably the cells are cultured at 37°C.

[0136] Suitably the cells are cultured at 5% CO2.

[0137] Suitably, RPMI-1640 may be from any source such as ThermoFisher Scientific (Fisher Scientific - UK Ltd, Bishop Meadow Road Loughborough, UK, LE11 5RG), Catalogue Number: 11875101.

[0138] In embodiments directed to the production of clinical grade materials (e.g. products intended for administration to human or animal subjects, suitably human subjects), suitably RPMI medium is replaced with X-VIVO™-10, a serum-free hematopoietic cell medium which contains L-glutamine, gentamicin and phenol red and is xeno-free. Suitably X-VIVO™-10 is available from Lonza Group Ltd, Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland, Catalogue #: BE04-380Q.

[0139] Alternatives to X-VIVO™-10 medium include TexMACS buffer, which needs to be supplemented with either GMP grade 5% human serum albumin (cat. no. 623160054 Biotest UK Ltd, 17 High Street, Longbridge, Birmingham, B31 2UQ, UK) or 5% human serum albumin (Albunorm™ cat. no. P / L 10673 / 0031, Octapharma Ltd, The Zenith Building, 26 Spring Gardens, Manchester M2 1AB, UK).

[0140] Further details can be found in the Examples section.

[0141] The cells of the present invention are not naturally occurring: they are produced solely by human intervention using the methods described herein.

[0142] It is possible that a very small / negligible number of triple positive cells may be present in the blood, but at very low (less than 1%) numbers, in which case such cells are barely detectable. Note that such cells may be shown to be present, but suitably do not express the 25F9 marker expressed by the cells of the present invention.

[0143] Suitably the cells of the invention are ex vivo.

[0144] Suitably the cell of the invention is in vitro.

[0145] Suitably, the cells of the invention are isolated. Suitably, isolated means removed or separated from at least one component of the cell's natural environment. For example, isolated may mean removed from the human or animal body. For example, isolated may mean separated from red blood cells (erythrocytes).

[0146] Suitably the cells of the invention are present in a population of cells.

[0147] Suitably, the invention provides a population of cells.

[0148] Suitably, the population of cells comprises more than 70% macrophages or monocytes.

[0149] Suitably, the population of cells comprises 0% red blood cells.

[0150] Suitably, the population of cells comprises a percentage of MSCs of 30% or less.

[0151] These are the release criteria for the final product during clinical trials.

[0152] Cells that are positive for one of the triple markers disclosed herein separately may have been observed in the prior art. However, the combination of triple positive markers has never been observed on cells such as macrophages. The inventors use the methods described herein to force cells to have the specific phenotype of the present invention.

[0153] Without wishing to be bound by theory, MRC1 / TIE2 double positive cells may be useful in treating patients as described herein. Thus, in a particular embodiment, macrophages or monocytes expressing the markers MRC1 and TIE2, respectively, are described. Thus, in a particular embodiment, treatment of patients using said double positive cells is described. Thus, in a particular embodiment, said double positive cells are described for use in treating diseases disclosed herein, such as CLI. However, most suitably, the cells used in the present invention are triple positive for MRC1, TIE2 and CD163.

[0154] Rybalko et al. 2017 (Regen. Med. Volume 12, number 2, pages 153-167) describes the therapeutic potential of adipose tissue derived stem cells and macrophages for ischemic skeletal muscle repair. The method used in Rybalko et al. would not produce triple expression of MRC1, TIE2 and CD163, or at least would not produce triple expression of MRC1, TIE2 and CD163 in any clinically useful amount. Rybalko et al. use a transwell approach in engineering their cells. The inventors assert that such an approach does not produce cells according to the invention. Rybalko et al. take more than 14 days to produce cells. There is no disclosure of triple positive MRC1 / TIE2 / CD163 positive cells in Rybalko et al.

[0155] More specifically, Rybalko et al. does not disclose any cell composition / cell population of the present invention. A very small number / very small percentage of triple positive cells as described herein may be present in a population of cells created by following the teachings of Rybalko et al. First, however, triple positive cells are present at a "very small" level, if at all, in the population of cells of Rybalko et al. For example, the inventors determine that approximately 0.3% or less of the cells of Rybalko et al. should be triple positive. With the knowledge of the present invention that triple positive cells are clinically useful, the best one can get by implementing the method of Rybalko et al., or by using this hindsight to try to maximize the number of triple positive cells available from the Rybalko method, is approximately 2% triple positive cells in the population of cells of Rybalko et al. In contrast, the inventors teach a method for generating a population of cells with at least about 50% triple positive cells. The maximum percentage of triple positive cells generated in Rybalko et al. is approximately 1-2%, which is comparable to the number of triple positive cells that would be generated if no treatment was applied, e.g., if the cells were simply cultured. Suitably, the cell composition of the present invention comprises at least about 50% triple positive cells. If any further evidence is needed, the inventors refer to the Examples section, which presents comparative data.

[0156] dose Suitably the invention provides a dose of cells.

[0157] In one embodiment, suitably the dose of cells is approximately 10e6 to 10e9 cells (i.e., 10 6 ~10 9 More appropriately, the dose is 10 6 ~10 9 In one embodiment, the dose comprises 10 6 ~10 9In one embodiment, suitably the dose of cells is approximately 10 6 ~10 9 In one embodiment, the dose of cells comprises approximately 10 cells, of which at least 50% are monocytes / macrophages, and more suitably at least 60% are monocytes / macrophages. 6 ~10 9 cells, among which at least 50% of the monocytes / macrophages present are triple positive monocytes / macrophages.

[0158] A suitable dose according to the present invention comprises approximately 100 million monocytes / macrophages per injection.

[0159] In one embodiment, suitably one dose is administered to a subject each week.

[0160] In one embodiment, suitably the course of treatment is for 10 weeks, ie 10 doses, one dose per week.

[0161] Most suitably, in one embodiment, the cell dose is suitably between about 1×10e8 and 2×10e8 cells (i.e., 1×10 8 ~2×10 8 cells, sometimes written as 1x10^8 to 2x10^8 cells), i.e. 100-200 million cells per dose. More suitably, the dose comprises 100-200 million monocytes / macrophages of the invention. In one embodiment, the dose comprises 100-200 million monocytes / macrophages of the invention. In one embodiment, suitably, the dose of cells comprises approximately 100-200 million cells, of which at least 50% are monocytes / macrophages, more suitably at least 60% are monocytes / macrophages. In one embodiment, the dose of cells comprises approximately 100-200 million cells, of which at least 50% of the monocytes / macrophages present are triple positive monocytes / macrophages.

[0162] In one embodiment, suitably one dose is administered to the subject every 6 to 12 weeks, most suitably every 12 weeks or every 3 months.

[0163] In one embodiment, suitably the course of treatment is 3 to 6 doses.

[0164] In one embodiment, suitably the course of treatment is 5 doses or 200 million cells per dose.

[0165] In one embodiment, the dose is suitably 2-3 million cells per kg of subject body weight. Thus, for an adult male weighing 75 kg, the dose is suitably 150-225 million cells, more suitably 150-200 million cells. Based on this information, it is routine for a clinician to calculate doses for other body weights or genders.

[0166] A dose may contain more than 100 million cells, for example, there may be a low level of stem cells from co-culture of macrophages of the present invention in the dose to be administered. For example, a dose may contain up to approximately 10% additional cells of the number of macrophages of the present invention contained in the dose. For example, if a dose contains 100 million macrophages of the present invention, the total number of cells in the dose may reach, for example, 110 million - 100 million macrophages according to the present invention plus an additional 10 million "other" cells, for example stem cells from the co-culture step in the production of macrophages of the present invention. In this example, the dose contains 90.9% macrophages of the present invention (100m macrophages of the present invention / 110m total cells=90.9%).

[0167] Suitably the dose comprises at least 50% of the cells of the invention; more suitably at least 60%; more suitably at least 65%; more suitably at least 69%; more suitably at least 70%; more suitably at least 80%; more suitably at least 85%; more suitably at least 90%; more suitably at least 95%; more suitably at least 96%; more suitably at least 97%; more suitably at least 98%; most suitably 99% or more of the cells according to the invention. In a preferred embodiment, suitably the dose comprises at least 98%, or at least 99% or more of the cells according to the invention, such as triple positive macrophages expressing MRC1, TIE2 and CD163.

[0168] In one embodiment, the cell numbers in the doses discussed herein refer to the number of triple positive monocyte / macrophage cells of the present invention.

[0169] With respect to cell preparation for dose manufacture, in a typical method of the invention, a total monocyte population of 2 billion is obtained from leukapheresis and used as the starting cells for the method of the invention, meaning that at the end of the co-culture, up to about 1-1.4 billion are triple positive. This allows for one or two jumbo doses of over 1 billion cells, or more appropriately multiple doses (e.g., 6 doses) of up to 1-200 million cells / dose.

[0170] Without wishing to be bound by theory, based on the inventors' 3% triple positive findings in CLI patients, any of the prior art techniques described herein using day 7 would generate 60 million cells - not even useful for a single dose.

[0171] number of cells A percentage of cells in reference to a cell type is suitably the percentage of an entire population of cells that are of the specified cell type. Thus, a figure of "at least 25% macrophage or monocyte cells" suitably means that 25% of the cells in an entire population of cells are macrophage or monocyte cells. For example, if there are 1000 cells in a sample, "25% macrophage or monocyte cells" means that 25% of the cells in an entire population of 1000 cells are macrophage or monocyte cells (i.e., 250 / 1000 are macrophage or monocyte cells).

[0172] The percentage of cells associated with marker expression is suitably the percentage of the population of monocytic cells expressing the cited marker. Thus, a figure of 50% "triple positive cells" suitably means that 50% of the monocytes are triple positive in the population of cells. For example, if there are 1000 cells in a sample, 50% "triple positive cells" means that 50% of the monocytes are triple positive in the 1000 cells. Thus, if there are 100 monocytes in a total number of 1000 cells, 50% "triple positive cells" means 50 triple positive monocytes (50 / 100 monocytes are triple positive) out of a total cell count of 1000. Suitably, the cells of the present invention comprise at least 50% "triple positive cells", i.e. suitably 50%, more suitably more than 50% triple positive cells (monocytes). It should be noted that references herein to "monocytes" may be replaced with references to "monocytes / macrophages" or "Mo / MΦ" or "CD14+ and / or CD45+ cells", e.g., "CD14+ bone marrow and / or CD45+ bone marrow cells" or "mononuclear cells taken from the blood of a subject", as will be apparent from the remainder of this document. The discussion of monocytes is a simplified representation to aid understanding and does not exclude or omit the other cell types described.

[0173] Suitably, the proportion of a population of cells produced according to the methods of the invention that are "monocytes / macrophages" (or "Mo / MΦ" or "CD14+ and / or CD45+ cells", e.g. "CD14+ bone marrow and / or CD45+ bone marrow cells" or "mononuclear cells taken from the blood of a subject") is at least 50%, more suitably at least 60%, more suitably at least 70%, more suitably at least 80%; more suitably at least 90%; more suitably at least 95%; more suitably at least 96%; more suitably at least 97%; more suitably at least 98%; most suitably 99% or more of the cells according to the invention. In other words, where the method produces a population of mononuclear cells taken from the subject's blood that have been co-cultured with MSCs, then after the co-culture is completed (and, if desired, after any purification / enrichment that may be performed on the mononuclear cells post-co-culture), suitably at least 50% of the cells present in the population are "monocytes / macrophages" (or "Mo / MΦ" or "CD14+ and / or CD45+ cells", e.g. "CD14+ bone marrow and / or CD45+ bone marrow cells" or "mononuclear cells taken from the subject's blood").

[0174] Suitably, the proportion of cells produced according to the method of the invention that express the triple positive marker is at least 30%, more suitably at least 40%, more suitably at least 50%, more suitably at least 60%; more suitably at least 65%; more suitably at least 69%; more suitably at least 70%; more suitably at least 80%, more suitably at least 85%; more suitably at least 90%; more suitably at least 95%; more suitably at least 96%; more suitably at least 97%; more suitably at least 98%; most suitably at least 99% or more of the cells according to the invention. In other words, if the method starts with an input of 1 billion mononuclear cells taken from the subject's blood, most suitably the method of the invention will convert at least (for example) approximately 80% of the mononuclear cells into triple-expressing mononuclear cells, i.e. 800 million triple-expressing cells from an input of 1 billion mononuclear cells.

[0175] Suitably, the mononuclear cells comprise macrophages or monocytes.

[0176] In one embodiment, suitably said mononuclear cells comprise monocytes.

[0177] More suitably, in one embodiment, said mononuclear cells comprise macrophages.

[0178] In a most suitable embodiment, the present invention relates to a cell population comprising at least 70% macrophage or monocyte cells (or "Mo / MΦ" or "CD14+ and / or CD45+ cells", e.g. "CD14+ bone marrow and / or CD45+ bone marrow cells" or "mononuclear cells taken from the blood of a subject"), and wherein at least 50% of said macrophages or monocyte cells express the marker: · MRC1; TIE2; and CD163 Each of the above is expressed.

[0179] Triple positive Suitably, a cell is said to express a marker discussed herein if said marker is detectable, for example using immunodetection to assay expression.

[0180] Suitably, where a cell is described as "triple positive", this means that the cell is positive for MRC1, TIE2 and CD163.

[0181] A cell is designated "triple positive" if each of the markers is detectable as described above.

[0182] The advancement taught by the present invention is that triple positive cells are clinically useful. It is possible that a very small portion of the population of cells in the prior art may contain the triple positive cells described herein, but there is no teaching in the art that such triple positive cells are clinically useful. Thus, in one embodiment, the present invention relates to the use of the triple positive cells described herein in medicine.

[0183] More Markers - 25F9 Suitably, the cells of the invention express human 25f9, a marker not present on circulating monocytes, which indicates that monocytes have matured into macrophages.

[0184] The cells begin to express 25F9 in culture as described above.

[0185] The cells express 25F9 on day 3 in the culture described above, indicating that the cells of the invention mature very rapidly in the presence of MSCs.

[0186] The cells of the present invention (triple positive cells that also express 25F9) do not naturally circulate in the blood, and this advantageous expression pattern is only produced by the manipulations taught herein, such as ex vivo manipulations, such as co-culture with MSCs.

[0187] 25F9 can be detected by any suitable method known in the art.

[0188] Most suitably, the detection of 25F9 is by the use of an affinity reagent such as an antibody (anti-25F9 antibody) that can specifically bind and detect 25F9 protein on the cell surface for cell expression. Suitably, the anti-25F9 antibody can be from any source.

[0189] Most suitably, detection of 25F9 is by use of the following antibody: (Mature Macrophage Marker Monoclonal Antibody (eBio25F9(25F9)), eFluor 660, eBioscience): Catalogue No. 50-0115-42, Thermo Fisher (Fisher Scientific - UK Ltd, Bishop Meadow Road Loughborough, UK, LE11 5RG). Suitably, detection is carried out according to the manufacturer's instructions.

[0190] Suitably, the antibody is Mouse / IgG1, kappa. The recommended isotype control is Mouse IgG1 kappa Isotype Control (P3.6.2.8.1), eFluor 660, eBioscience. Suitably, the antibody is stored in PBS, pH 7.2, containing 0.1% gelatin, 0.2% BSA. Optionally, 0.09% sodium azide. Do not freeze. Store in the dark at 4°C.

[0191] Monoclonal antibody 25F9 recognizes a protein on mature macrophages both on the cell surface and within intracellular vesicular structures. No expression is found on immature macrophages or monocytes or any other hematopoietic cells. Reported Uses: This eBio25F9 (25F9) antibody has been reported for use in flow cytometric analysis. Tested Uses: This eBio25F9 (25F9) antibody has been previously titrated and tested by flow cytometric analysis of cultured human macrophages derived from monocytes. It can be used at 5 μL (0.25 μg) per test. A test is defined as the amount (μg) of antibody that will stain a cell sample in a final volume of 100 μL. Cell numbers must be empirically determined but can range from 10^5 to 10^8 cells / test. eFluor® 660 is an alternative to Alexa Fluor® 647. eFluor® 660 emits at 659 nm and is excited with a red laser (633 nm). Please make sure your instrument is capable of detecting this fluorochrome. Excitation: 633-647 nm; Emission: 668 nm; Laser: Red laser. Filtration: Post-manufacture filter 0.2 μm.

[0192] Suitably, at least 50% of the monocyte / macrophage cells of the present invention are triple positive and express 25F9. We typically observe that at least 80% of the cells of the present invention express 25F9. Thus, referring to FIG. 11B, 80% of 69.96% triple positive cells = 55.968% (triple positive + 25F9) of the cells in the preparation are (triple positive + 25F9). We refer to FIG. 24, which shows that there is a significant increase in the expression of 25F9 in MSC-primed monocytes compared to monocytes cultured alone.

[0193] Medical Indications The present invention finds application in peripheral vascular disease.

[0194] The present invention finds application in peripheral arterial disease.

[0195] The present invention finds application in critical limb ischemia.

[0196] The present invention finds application in fibrosis.

[0197] In one embodiment, the present invention finds application in the treatment of fibrosis, such as pulmonary fibrosis. In one embodiment, suitably, the subject has COVID-19 or has previously had COVID-19. This is particularly beneficial because COVID-19 patients may have long-term lung problems.

[0198] The present invention finds application in ischemic stroke.

[0199] In one embodiment, suitably, the subject has asymptomatic Peripheral Arterial Disease (PAD). Thus, the present invention may be for use in treating Peripheral Arterial Disease (PAD). In one embodiment, the present invention provides a method for treating Peripheral Arterial Disease (PAD), comprising administering to a subject the cells and / or compositions of the present invention.

[0200] In one embodiment, suitably, the subject suffers from intermittent claudication. Thus, the present invention may be for use in treating intermittent claudication. In one embodiment, the present invention provides a method for treating intermittent claudication, comprising administering to a subject the cells and / or compositions of the present invention.

[0201] In one embodiment, suitably, the subject has worsening claudication. Thus, the present invention may be for use in treating worsening claudication. In one embodiment, the present invention provides a method for treating worsening claudication, comprising administering the cell and / or composition of the present invention to a subject.

[0202] The cells and / or compositions of the invention may be for use in the treatment of critical limb ischemia or comprehensive severe chronic limb ischemia (CLTI). In one embodiment, the invention provides a method for treating critical limb ischemia or comprehensive severe chronic limb ischemia (CLTI), comprising administering the cells and / or compositions of the invention to a subject.

[0203] The cells and / or compositions of the invention may be for use in the treatment of fibrosis. The cells and / or compositions of the invention may be for use in the treatment of fibrosis, such as pulmonary fibrosis. In one embodiment, the invention provides a method of treating fibrosis comprising administering the cells and / or compositions of the invention to a subject. In one embodiment, the invention provides a method of treating fibrosis comprising administering the cells and / or compositions of the invention to a subject who has or has previously had COVID-19, for example, for use in the treatment of COVID-19 induced pulmonary fibrosis.

[0204] The cells and / or compositions of the invention may be for use in the treatment of other forms of interstitial lung disease, such as idiopathic pulmonary fibrosis.

[0205] The cells and / or compositions of the invention may be for use in the treatment of stroke, suitably ischemic stroke, hi one embodiment, the invention provides a method of treating stroke comprising administering the cells and / or compositions of the invention to a subject.

[0206] The cells and / or compositions of the present invention can be for use in post-ischemic revascularization. More specifically, post-ischemic revascularization of the heart, brain or leg. In one embodiment, the present invention provides a method for inducing post-ischemic revascularization, comprising administering the cells and / or compositions of the present invention to a subject.

[0207] The cells and / or compositions of the invention may be for use in inducing blood vessel growth, e.g., angiogenesis and / or arteriogenesis. In one embodiment, the invention provides a method of inducing angiogenesis and / or arteriogenesis comprising administering the cells and / or compositions of the invention to a subject.

[0208] The cells and / or compositions of the invention may be for use in stimulating the production of new blood vessels in a mammal.

[0209] The cells and / or compositions of the invention may be for use in stabilizing blood vessels. In one embodiment, the invention provides a method of stabilizing blood vessels, comprising administering the cells and / or compositions of the invention to a subject.

[0210] The cells and / or compositions described herein are useful in treating fibrosis. Thus, the compositions of the present invention can be used to treat fibrosis.

[0211] The fibrosis may be renal fibrosis, liver fibrosis, cardiac fibrosis and / or pulmonary fibrosis.

[0212] We disclose that the triple positive cells described herein produce hepatocyte growth factor (HGF) and / or IL-10 and TNFα, which are anti-fibrotic proteins. This is a further advantage of the cell composition of the present invention. The cells of the present invention also produce higher levels of IL-10 and TNFα, but not HGF, in response to inflammatory stimulation by lipopolysaccharide (LPS). We refer to the figures.

[0213] Suitably administration is intramuscular, for example into an ischemic limb of a patient with peripheral vascular disease, such as peripheral arterial disease (eg CLI).

[0214] The present invention can be used as an adjunct therapy in subjects who have had revascularization of their proximal inflow vessels.

[0215] The present invention can be used as a stand-alone treatment for subjects who are poor candidates for surgery.

[0216] The present invention may also be used intraoperatively in patients having endovascular revascularization, where primed cells may be delivered directly to a terminal arteriole at the site of a known vascular occlusion.

[0217] The present invention may also be used for the treatment of other cardiovascular diseases where tissue ischemia is present, including patients with ischemic heart disease, heart failure and / or patients with cerebrovascular disease (eg stroke).

[0218] Cell Production In one embodiment the cells / populations of cells of the invention may be produced by providing a total monocyte population isolated from a patient (suitably having been previously isolated, i.e. in one embodiment suitably the step of providing a population of monocytes comprises the step of providing an in vitro population of monocytes).

[0219] Suitably, the monocytes are obtained by leukapheresis, which has the advantage of providing high cell numbers.

[0220] Suitably, monocytes are obtained from a single blood donation (eg up to 485ml), which has the advantage of not requiring a leukapheresis machine.

[0221] Suitably the monocytes / macrophages are human monocytes / macrophages.

[0222] How to Isolate Monocytes / Macrophages Monocytes / macrophages can be separated according to their expression of CD14. These cells are - by labeling with immunomagnetic anti-CD14 beads and separating the cells by passing them over a magnetic column; or - Can be fluorescently stained with CD14 and then removed using fluorescence activated cell sorting.

[0223] Suitably the monocytes are then primed with MSCs.

[0224] Suitably the MSCs are human MSCs.

[0225] The MSCs may be from any suitable source, for example either from bone marrow or from adipose tissue (suitably having been previously separated, i.e. in one embodiment suitably the step of providing MSCs comprises the step of providing an in vitro population of MSCs).

[0226] MSCs from a range of sources may be used in the present invention, for example commercially available MSCs and / or GMP compliant MSCs may be used in the present invention.

[0227] MSC banks can be generated from adipose tissue or bone marrow of healthy human subjects using known methods.

[0228] MSCs from various sources can be used. Suitably, MSCs are from RoosterBio Inc. (5295 Westview Drive, Suite 275, Frederick, MD 21703, USA) and cultured in RoosterBio Inc.'s medium (RoosterNourish-MSC-CC, KT-021), following the manufacturer's protocol for culturing MSCs. Other commercially available MSCs and / or media can also be used, e.g. MSCs from NHSBT (NHS Blood and Transplant, 500 North Bristol Park, Filton, Bristol, BS34 7QH, UK) and / or media from Miltenyi Biotec (MSC-Brew, 170-076-325, Miltenyi Biotec Ltd., Almac House, Church Lane, Bisley, Surrey GU24 9DR, UK) and / or Sartorius (MSC NutriStem XF Medium, 05-200-1A - Sartorius, Otto-Brenner-Strasse 20, 37079 Goettingen, Germany).

[0229] We refer to Example 10 if any further guidance is needed.

[0230] The priming step (sometimes referred to as the "education" step) of the present invention suitably comprises co-culturing monocytes and MSCs.

[0231] The resulting primed monocytic cells are then analyzed to confirm successful transformation of their phenotype. The cells may then be stored. These cells can then be used to administer to a subject over a course of several weeks as a course of treatment and / or as a one-time administration, e.g., injection.

[0232] advantage Prior art attempts to isolate functional cells have involved "superselection", i.e. procedures designed to select cells present at very low frequencies in the starting population. However, the number of cells obtained from such "superselection" methods is very low, which may be due to the fact that the frequency of these cells in the starting population is extremely low. This is a problem in the art. An advantage of the present invention is that a very high efficiency of conversion of the starting cells to the functional triple positive cells of the invention is achieved. An advantage of such an efficient conversion is that a very high percentage of the cells of the invention are produced from the starting population. An additional advantage is that a much higher absolute number of cells can be easily produced than by using the uncertain "superselection" methods known in the art.

[0233] Thus, in one aspect the invention relates to a mixture of cells, said mixture comprising at least 50%, more suitably at least 80%, macrophages expressing MRC1, TIE2 and CD163.

[0234] Method Steps Suitably the starting cells comprise or consist of macrophages or monocytes. Most suitably the starting cells comprise or consist of monocytes.

[0235] Where the starting cells are part of a population of cells, for example a population of mononuclear cells previously obtained from a blood sample, suitably said population of cells comprises at least 20% macrophages or monocytes, most suitably monocytes.

[0236] Suitably, said starting cells comprise or consist of PBMCs.

[0237] Suitably, said macrophages or monocytes are derived from PBMCs.

[0238] Macrophages or monocytes may be separated from PBMCs by any suitable method known in the art, such as immunomagnetic bead separation or fluorescence activated cell sorting, suitably using anti-CD14 or anti-CD45, more suitably anti-CD14.

[0239] The population of cells may be enriched for macrophages or monocytes by any suitable method known in the art, such as immunomagnetic bead separation or fluorescence activated cell sorting, suitably using anti-CD14 or anti-CD45, more suitably anti-CD14.

[0240] Culture time In the methods of the present invention, the starting cells are monocytes, such as primary monocytes.

[0241] The present invention does not include any method that starts with macrophages (which may technically have been blood-derived, but which have in fact already been cultured in vitro for some sustained period of time to differentiate into macrophages).

[0242] Primary monocytes are defined as CD14 expressing monocytes that have been isolated from blood and have not yet been placed in culture (i.e., are still in suspension). As soon as monocytes are placed in a culture dish, they become fixed (adherent) and slowly begin to differentiate into macrophages, so they are no longer primary monocytes. Monocytes that are incubated in vitro were primary monocytes before they were cultured.

[0243] The method of the present invention involves directly exposing primary monocytes to MSCs on day 0, ie, culturing / contacting primary monocytes with MSCs directly on day 0.

[0244] Culture times / days are counted from the time primary monocytes are placed into culture.

[0245] Our cells are placed into culture immediately after isolation - the start time is the time the cells are placed into culture. For example, if cells are isolated on Monday AM and then placed into culture at 11am, then Tuesday at 11am is the 24 hours (day 1) of culture.

[0246] Cells can be harvested and stored in cryogenic conditions (by freezing) and when thawed, the cells remain viable.

[0247] Release Criteria It should be noted that cells from different patients exhibit different levels of plasticity and / or different rates of change. Thus, the amount of time required in culture to induce the desired markers may be longer for patients who are older or who exhibit other characteristics that affect the rate of change in their mononuclear blood cell expression. Thus, in one embodiment, suitably, the method of the invention involves a "release" step. In this embodiment, cells are harvested ("released") only if certain predefined criteria are met. For example, cells can be maintained in culture, samples of those cells can be taken (or samples can be taken that have been cultured separately in smaller "sibling" cultures with the same proportions), tested, and the cells can be released to the next step of the method only if they meet a predefined value, such as the proportion of cells expressing the triple markers MRC1, TIE2, and CD163. It should be noted that in clinical trial data, less than three markers may be assayed, for example, only MRC1 and CD163 may be assayed. This is the "release assay". This is a shortened assay used to ensure speed and reproducibility towards release criteria in clinical practice (MHRA approved release assay). This release assay does not mean that the cells are positive for only two of the three specified markers - in fact the cells we describe are positive for all three markers as claimed - this is merely an accepted practice in the field that has been recommended / approved by the MHRA to create an efficient and rapid "release assay" that can be used to test as well as release clinical products - this should be distinguished from performing scientifically rigorous testing to demonstrate expression of all three markers as shown throughout this document. A release assay is a shorthand for confirming that the product (the cells of the invention) is ready for clinical release - this is a different criterion than the scientific demonstration of expression of all three markers which still applies for the cells of the invention.

[0248] Notwithstanding this, suitably the present invention requires culture for no more than 7 days, more suitably no more than 5 days.

[0249] An advantage of the present invention is that an autologous cell product is provided.

[0250] It will be noted that macrophages / monocytes do not actually proliferate in culture. Thus, in one aspect, the method of the invention may comprise: Collection of macrophage cells by leukapheresis or whole blood donation Start the culture with approximately 2 billion monocyte / macrophage cells (leukapheresis) or up to approximately 500 million monocyte / macrophage cells (whole blood donation) Co-culturing mesenchymal stem cells and macrophage cells Evaluate phenotypic changes and harvest when at least 50% and more appropriately at least 80% of macrophage cells express MRC1 and TIE2 and CD163.

[0251] Medical Methods In one embodiment, suitably the method comprises isolating macrophages from mesenchymal stem cells co-cultured with macrophages.

[0252] Suitably, the macrophages are administered to a subject in need thereof.

[0253] Free Cells and Encapsulation In one embodiment, the triple positive cells of the present invention are injected into a patient, which may be referred to as "free cells."

[0254] In one embodiment, the triple positive cells of the present invention are encapsulated and injected into a patient in the encapsulated state.

[0255] Suitably the compositions of the invention are encapsulated, this has the advantage of improving retention and / or overcoming "washout", whereby cells are lost from the injection site over time.

[0256] Suitably the cells may be encapsulated using any suitable method known in the art.

[0257] An advantage of the present invention is that the desired cells retain their triple positive characteristics when encapsulated, as demonstrated in Example 4.

[0258] In one embodiment, the cells are cultured for 3 to 7 days, more suitably 3 days, and then reintroduced into the patient.

[0259] In one embodiment, the cells are immediately encapsulated, the encapsulated cells are cultured, for example for 3 to 7 days, more suitably 3 days, and then the encapsulated cells are reintroduced into the patient.

[0260] In this embodiment, triple positive cells may not be separated from MSCs that are co-cultured with them to induce triple positive expression. Thus, in one embodiment, monocytes / macrophages and MSCs may be co-encapsulated. More appropriately, monocytes / macrophages and MSCs are co-encapsulated at a ratio of 3:1. To the best of our knowledge, this is the first disclosure of encapsulating monocytes / macrophages and MSCs together in a single encapsulation.

[0261] In one embodiment, suitably, monocytes / macrophages are encapsulated. In one embodiment, the present invention provides an encapsulation in which triple positive monocyte / macrophage cells are contained. In one embodiment, said encapsulation further comprises MSCs. When the encapsulation comprises both triple positive monocyte / macrophage cells and MSCs, suitably, they are present in a ratio of monocyte / macrophage cells:MSCs of 3:1. Most suitably, they are present in a ratio of triple positive monocyte / macrophage cells:MSCs of 3:1.

[0262] Negative markers Suitably, the cells produced according to the methods of the present invention detect the following markers: ·MMP-9 (Consignment: P14780) NRP1 (Accession No. 014786 - also known as CD304) ·HB-EGF (Consignment: P01133) is down-regulated (i.e., lower expression compared to primary monocytes cultured alone and / or compared to primary monocytes that are not cultured).

[0263] Regarding MMP-9, this marker is downregulated when co-cultured with MSCs, but is also upregulated when monocytes are cultured without MSCs.

[0264] In one embodiment, if a particular marker is undetectable, the cell is said to not express said marker.

[0265] The detection / assessment of downregulation is suitably assessed using mean fluorescence intensity, i.e. the amount of protein expressed. Suitably, this is expressed as a percentage of the level measured for primary monocytes, i.e. monocytes isolated from peripheral blood that have not been cultured in vitro / ex vivo. A protein is considered to be downregulated if it is expressed (detected) at a level lower than that for primary monocytes.

[0266] Suitably, a protein is considered to be downregulated when it is expressed (detected) at a statistically significant level lower than the level for primary monocytes.For MMP-9, suitably, there is a median 60% decrease in expression when monocytes are primed with MSCs.The inventors have performed further statistical analysis to show that 50% of cells will downregulate MMP-9 by at least this 60% value when co-cultured with MSCs.For HB-EGF, suitably, there is a 30% decrease in the expression of this protein when the inventors co-culture monocytes with MSCs.Suitably, 15% of cells will be downregulated at least 30% for HB-EGF each time.

[0267] Sequence identity When assessing expression of the markers described herein, detection of a marker can be found when the exact sequence has been specified or when the desired gene / protein is known to be expressed. For example, allelic variants or normal genetic variation between individuals within a species is a well-known phenomenon, and minor or negligible sequence differences between (for example) a marker of interest and the reference sequence being assessed will not affect the assessment of whether the marker is expressed or not in a sample / cell.

[0268] It may be desirable to consider sequence relationships in terms of sequence identity.

[0269] Comparison of sequences can be performed by eye, or more usually, with the aid of readily available sequence comparison programs. These publicly and commercially available computer programs can calculate the percent homology (such as percent identity) between two or more sequences.

[0270] Percent identity can be calculated over consecutive sequences, i.e., one sequence is aligned with the other sequence, and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, one residue at a time. This is called "ungapped" alignment. Usually, such ungapped alignment is only performed over a relatively short number of residues (e.g., less than 50 consecutive amino acids).

[0271] Although this is a very simple and consistent method, it fails to take into account that, for example, in an otherwise identical sequence pair, a single insertion or deletion can cause subsequent amino acid residues to be unable to be aligned, thus resulting in a large drop in the percent homology (percent identity) when a global alignment is performed. As a result, most sequence comparison methods are designed to produce optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology (identity) score. This is achieved by inserting "gaps" in the sequence alignment to maximize local homology / identity.

[0272] These more sophisticated methods assign a "gap penalty" to each gap present in the alignment, so that for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting a higher relatedness between the two compared sequences - will result in a score higher than 1 with many gaps. An "affine gap cost" is usually used, which imposes a relatively high cost on the presence of a gap and a smaller penalty on each subsequent residue in the gap. This is the most commonly used gap scoring system. Higher gap penalties will, of course, produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified; however, it is preferred to use the default values ​​when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package (see below), the default gap penalties for amino acid sequences are -12 for a gap and -4 for each extension.

[0273] Therefore, the calculation of maximum homology percentage first requires the creation of an optimal alignment that incorporates gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software that can perform sequence comparisons include, but are not limited to, the BLAST package, FASTA (Altschul et al., 1990, J. Mol. Biol. 215:403-410) and the comparison tool of the GENEWORKS suite.

[0274] Although the final percentage homology can be measured in terms of identity, the alignment process itself is not usually based on an all-or-nothing pairwise comparison. Instead, a scaled similarity score matrix is ​​typically used that assigns a score to each pairwise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs typically use either the public default values ​​or a custom symbol comparison table, if supplied. It is preferred to use the public default values ​​for the GCG package, or a default matrix such as BLOSUM62 for other software. Once the software has produced an optimal alignment, it is possible to calculate the percentage homology, preferably the percentage sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.

[0275] Reference sequence / database release Sequences deposited in a database may change over time. Suitably, reliance is placed on the current version of the sequence database(s). Alternatively, reliance is placed on the release in effect on the filing date.

[0276] As one of skill in the art would know, the accession number may be a version / date accession number. Citable accession numbers for current database entries are the same as herein, but omitting the decimal point and any digits following it.

[0277] GenBank is the NIH genetic sequence database, an annotated collection of all publicly available DNA sequences (National Center for Biotechnology Information, US National Library of Medicine 8600 Rockville Pike, Bethesda MD, 20894 USA; Nucleic Acids Research, 2013 Jan;41(D1):D36-42), to which the accession numbers provided relate unless otherwise clear. Where appropriate, the current release is relied upon. More appropriately, the release available at the effective filing date is relied upon. Most appropriately, the GenBank database release referenced is NCBI-GenBank Release 241: December 15, 2020.

[0278] Where appropriate, reliance is placed on the current version of the sequence database. Alternatively, reliance is placed on the release in effect on the filing date. For the avoidance of doubt, reliance is placed on the UniProt consortium European Bioinformatics Institute (EBI), SIB Swiss Institute of Bioinformatics and Protein Information Resource (PIR)'s UniProt Knowledgebase (UniProtKB) Release 2021_01, published on February 10, 2021. UniProt (Universal Protein Resource) is a comprehensive catalog of information about proteins ("UniProt: the universal protein knowledgebase" Nucleic Acids Res. 45: D158-D169 (2017)).

[0279] Further Applications The properties of the primed Mo / MΦ of the present invention may be affected by cryopreservation. The data that the present inventors have indicates that the viability of the Mo / MΦ of the present invention is not affected after cryopreservation and storage. Those skilled in the art can monitor whether the effectiveness of the cells of the present invention in revascularizing ischemic tissue changes after storage, and if necessary, the storage and / or the dose of the cells administered to the subject may be changed accordingly.

[0280] Suitably, the cells / populations of cells of the present invention are produced in a GMP facility.

[0281] The present invention may be used in the treatment of peripheral vascular disease and / or peripheral arterial disease, suitably in patients with intermittent claudication and / or global severe chronic limb ischemia. In this context, the cells of the invention stimulate the sprouting of new blood vessels (angiogenesis) as well as induce active remodeling of existing blood vessels (arteriogenesis).

[0282] The present invention can be used as a tissue remodeling, anti-fibrotic therapy to treat conditions associated with tissue fibrosis, including, for example, fibrotic lung, kidney, liver and skin diseases.

[0283] The present invention can be used to treat ischemic heart disease or heart failure as a consequence of fibrosis.In the case of ischemic heart disease, the present invention can be used to stimulate angiogenesis and / or arteriogenesis.In the case of heart failure as a consequence of fibrosis, the present invention can be used to induce active tissue remodeling, reduce fibrosis and / or improve myocardial function.

[0284] The present invention can be used to treat ischemic stroke, where the present invention induces the growth of new blood vessels via angiogenesis or arteriogenesis.

[0285] The present invention can be used to treat ischemic tissue, such as ischemic cardiac tissue or ischemic brain tissue.

[0286] Proof of effectiveness The present invention can be used in the treatment of peripheral vascular disease, suitably peripheral arterial disease, suitably CLI or global severe chronic limb ischemia (CLTI). The data in the present application support such use, for example, Figures 15A-D show significantly enhanced smooth muscle cell proliferation when cells are exposed to conditioned medium generated from the present invention, and Figures 16A,B show significantly improved revascularization after delivery of the present invention, along with evidence of arteriogenesis in vivo (Figures 16C,D). Thus, the reader will understand from the demonstration of smooth muscle proliferation in vitro and improved limb perfusion after delivery of the present invention to an ischemic limb in mice, and the greater number of blood vessels generated seen in vivo, that the present invention addresses ischemia, as these features result in improved blood supply to the ischemic tissue.

[0287] The present invention can be used in the treatment of fibrosis. Data in the present application support this use, for example, FIG. 19 shows that the present invention produces higher levels of the anti-fibrotic proteins HGF, IL-10 and TNFα compared to unprimed monocytes. FIG. 20 shows that the present invention reverses fibrosis in vitro via the reduction of SMA and fibronectin expression on stimulated fibroblasts. FIG. 21A shows that the present invention protects small airway epithelial cells from undergoing apoptosis, and FIG. 21B shows that the present invention improves endothelial proliferation. FIG. 22 shows the reduction of fibrosis in vitro (collagen staining) when the present invention is delivered to tissue after injury. Thus, what the reader will understand from the demonstration of the reduction of fibrosis in vitro and in vivo, and the improvement of survival of small airway epithelial and endothelial cells (during the fibrotic process, epithelial and endothelial damage and death occurs) is that the present invention will address fibrosis by reducing the fibroblast to mesenchymal transition and by improving survival of epithelial and endothelial cells.

[0288] The present invention can be used in inducing revascularization. The data in this application support this use, for example, FIG. 16A shows improved limb perfusion in mice treated with the present invention after induction of hindlimb ischemia; and FIG. 16C and D show that the muscle contains larger arterioles, suggesting vascular remodeling and arteriogenesis. The present invention also produces higher levels of HGF than cells cultured alone (FIG. 19), a protein known to stimulate revascularization of ischemic tissue. Thus, the reader will understand from the demonstration of improved limb perfusion and arteriogenesis that the present invention will induce revascularization, as angiogenesis and / or arteriogenesis are shown, and these processes directly lead to revascularization of ischemic tissue through inducing better collateral circulation.

[0289] The present invention can be used in the treatment of ischemic stroke. The data in this application supports this use, for example, Figures 15 and 16 show the improvement of in vitro smooth muscle cell proliferation and in vivo arteriogenesis, respectively. In addition, Figure 21B shows the improvement of vascular endothelial cell proliferation (angiogenesis). Therefore, what the reader will understand from the demonstration of the improvement of angiogenesis and arteriogenesis is that the present invention will address ischemic stroke, since new blood vessel formation in the ischemic brain allows reperfusion of the previously ischemic tissue from the stroke.

[0290] Further embodiments In one embodiment, the invention provides a method of treating a mammalian subject comprising administering to said subject a cell as described above.

[0291] In one embodiment, the invention provides a method of treating a mammalian subject comprising administering to said subject a population of cells as described above.

[0292] In one embodiment, the present invention provides mesenchymal stem cell-priming monocytes for tissue remodeling.

[0293] Further particular and preferred aspects are set out in the accompanying independent and dependent claims. Features of the dependent claims may be combined with features of the independent claims as appropriate in combinations other than those explicitly set out in the claims.

[0294] It will be understood that where features of an apparatus are described as operable to provide a function, the features of the apparatus include features of an apparatus that provide that function or that are adapted or configured to provide that function.

[0295] The invention will now be described through numbered paragraphs: Paragraph 1. A population of cells comprising at least 50% macrophage or monocyte cells, wherein at least 50% of said macrophage or monocyte cells express each of the markers: MRC1; TIE2; and CD163.

[0296] Paragraph 2. A population of cells according to Paragraph 1, comprising at least 70% macrophage or monocyte cells.

[0297] Paragraph 3. A population of cells according to Paragraph 1 or Paragraph 2, comprising at least 80% macrophage or monocyte cells.

[0298] Paragraph 4. A population of cells according to any of Paragraphs 1 to 3, wherein at least 60% of the macrophage or monocyte cells express each of the markers: MRC1; TIE2; and CD163.

[0299] Paragraph 5. A population of cells according to any of Paragraphs 1 to 4, wherein the macrophage or monocyte cells express human 25f9.

[0300] Paragraph 6. A population of cells according to any of Paragraphs 1 to 5, wherein the macrophage or monocyte cells express CD14 and / or CD45.

[0301] Paragraph 7. A population of cells according to any of Paragraphs 1 to 6, comprising a ratio of monocyte / macrophage cells:MSCs of 3:1.

[0302] Paragraph 8. A population of cells according to Paragraph 7, comprising triple positive monocyte / macrophage cells:MSCs in a ratio of 3:1.

[0303] Paragraph 9. A population of cells according to any of Paragraphs 1 to 8, wherein the cells are encapsulated.

[0304] Paragraph 10. A method comprising the steps of: (a) providing macrophages or monocytes from a subject; (b) providing MSCs; and (c) culturing said macrophages or monocytes with said MSCs.

[0305] Paragraph 11. The method of Paragraph 10, wherein step (c) comprises contacting macrophages or monocytes with MSCs to produce a cell mixture, and culturing the cell mixture.

[0306] Paragraph 12. A method according to Paragraph 10 or Paragraph 11, wherein in step (c) the ratio of (macrophages or monocytes):(MSCs) is 3:1.

[0307] Paragraph 13. The method of any of Paragraphs 10 to 12, wherein the cells are cultured for about 3 to 7 days, preferably about 3 to 5 days.

[0308] Paragraph 14. The method of Paragraph 13, wherein the cells are cultured for about 3 days.

[0309] Paragraph 15. The method of any of Paragraphs 10 to 13, wherein the cells are cultured in a medium, and the medium is changed every 5 days.

[0310] Paragraph 16. A population of cells according to any of Paragraphs 1 to 9 for use in the treatment of peripheral vascular disease, suitably global severe chronic limb ischemia.

[0311] Paragraph 17. A population of cells according to any of Paragraphs 1 to 9 for use in the treatment of fibrosis.

[0312] Paragraph 18. A population of cells according to any of Paragraphs 1 to 9 for use in the treatment of ischemic stroke.

[0313] Paragraph 19. A method of treating a mammalian subject comprising administering to said subject a population of cells according to any of Paragraphs 1 to 9.

[0314] Paragraph 20. About 10 6 ~10 9 20. The method of paragraph 19, comprising administering a dose of 100 million cells.

[0315] Paragraph 21. Use of a population of cells according to any of Paragraphs 1 to 9 for inducing angiogenesis in a mammal. [Brief description of the drawings]

[0316] The present invention will now be further described with reference to the accompanying drawings, in which: [Figure 1] FIG. 1 shows comparative data: despite increased expression of CD206, there is no increased expression of TIE2 or CD163 using the method of Rybalko et al. 2017. [Diagram 2] FIG. 2 shows a diagram of marker expression in MSC-primed monocytes (cells / cell populations according to the invention - see (B)) compared to control cells (not according to the invention - see (A)). [Diagram 3] FIG. [Figure 4] FIG. [Diagram 5] FIG. [Figure 6] FIG. 1 shows plots and graphs. [Figure 7] FIG. 1 shows plots and graphs. [Figure 8] FIG. 1 is a diagram showing a table (heat map). [Figure 9] FIG. [Figure 10] FIG. [Figure 11] Venn diagram. Numbers in Figure 11B are Mo / MΦ expression rates - 69.96% triple positive - n=21. [Figure 12] FIG. [Figure 13] FIG. [Figure 14] FIG. 1 is a diagram showing a Venn diagram. [Figure 15] FIG. 1 shows a graph and a bar graph. [Figure 16] FIG. 1 shows graphs, photographs and plots. [Figure 17] FIG. [Figure 18] FIG. [Figure 19] FIG. [Figure 20] FIG. [Figure 21] FIG. 1 shows plots and graphs. [Figure 22] FIG. [Figure 23] Figure 1 shows a graph: The top ("100") line is the data - 100% of mice were alive at all time points. [Figure 24] FIG. [Diagram 25] FIG. [Figure 26] FIG. [Figure 27] Figure 1 shows plots: Cells of the invention. n=7 / group (A) Day 3: No difference in IL-12 expression (IL-12 is still highly expressed on our cells after co-culture) (B) Day 7: No difference in IL-12 expression (IL-12 is still highly expressed on our cells after co-culture). [Figure 28] FIG. 1 shows a bar graph. TIE2 expression is highest on day 3 of coculture. TIE2 expression is significantly enhanced on day 3 and begins to decline on day 7. TIE2 then continues to decline to levels similar to control cells (P<0.0001 using Kruskal-Wallis test with post-hoc Dunn's multiple comparison text, n=6 samples, error bars are SEM). [Figure 29] FIG. 1 shows that CD206 expression is highest on day 3 of co-culture. CD206 expression is significantly enhanced on day 3 and begins to decline on day 7. CD206 then continues to decline to levels similar to control cells (P<0.0001 using Kruskal-Wallis test with post-hoc Dunn's multiple comparison text, n=6 samples, error bars are SEM). [Diagram 30] FIG. 1 shows that CD163 expression is highest on day 3 of co-culture. CD163 expression is significantly enhanced on day 3 and begins to decline on day 7. CD163 then continues to decline to levels similar to control cells (P<0.0001 using Kruskal-Wallis test with post-hoc Dunn's multiple comparison text, n=6 samples, error bars are SEM). [Diagram 31] Figures (i, ii, v, vi) and photographs (iii, iv) show results from a human study demonstrating the efficacy of the present invention in human subjects. For more details, the inventors refer to Example 15. [Examples]

[0317] Although exemplary embodiments of the present invention have been disclosed in detail herein with reference to the accompanying drawings, the present invention is not limited to such precise embodiments, and those skilled in the art may make various changes and modifications therein without departing from the scope of the present invention as defined by the appended claims and their equivalents. Example 1

[0318] Comparison Data Rybalko et al 2017 (Regen. Med. Volume 12, number 2, pages 153-167) showed enhanced CD206 expression on U-937 cells when co-cultured with bone marrow-derived MSCs. We compared our method of priming monocytes with their method by following their methodology.

[0319] U-937 cells (CRL-1593.2, ATCC) were cultured in RPMI-1650 growth medium supplemented with 10% FCS and 1% penicillin-streptomycin. For co-culture experiments, MSCs were grown in 6-well transwell inserts at 20,000 cells / cm. 2 U-937 cells were treated with 100 nM 12-O-tetradecanoylphorbol-13-acetate (TPA) for 48 h and seeded in 6-well plates at a concentration of 400,000 cells / well in RPMI medium containing 10% FCS. Following overnight culture, the medium was replaced with serum-free DMEM or RPMI medium / 10% FCS for stem cells and U-937 cells, respectively. The transwell inserts containing MSCs were transferred to the 6-well plates containing U-937 cells. For direct co-culture experiments, U-937 cells were treated with 100 nM TPA for 48 h and seeded in 6-well plates at a concentration of 400,000 cells / well in RPMI / 10% FBS. MSCs were then added at 1 × 10 5 Transwell and direct cultured U-937 cells were analyzed by flow cytometry.

[0320] The above methods confirmed that when U-937 cells were directly co-cultured and transwell co-cultured on MSCs, CD206 expression was increased to 67.6% and 69.2%, respectively, thus reproducing the data / methods disclosed in Rybalko et al.

[0321] However, with this method, TIE2 expression was only 3.1% and 0.7%, and CD163 expression was only 18.1% and 5.5%, in direct coculture and transwell coculture, respectively (Figure 1).

[0322] This resulted in "triple positive" expression of only 1% and 2% for direct and transwell co-cultured cells, respectively.

[0323] Thus, these comparative data indicate that the methods disclosed in Rybalko et al. do not generate the cells / cell populations of the present invention. Example 2

[0324] Antifibrosis experiments In one embodiment, the present invention provides the use of the cell composition described herein in the treatment of fibrosis. The data presented herein shows excellent efficacy in leg model. Those skilled in the art would expect this to make it realistic and convincing that the treatment works in lung fibrosis. In fact, based on the evidence provided in this document, MHRA (Medicines and Healthcare Products Regulatory Agency of the UK government) has proceeded with clinical application in lung. This illustrates that the exemplary data provided herein supports the use of the cell composition described in a range of anti-fibrotic clinical applications.

[0325] MSC-primed monocytes have anti-fibrotic activity in vitro. These cells (conditioned medium) stimulate a significant suppression of SMA expression (Figure 20A) and fibronectin (Figure 20B) expression (fibrosis markers) after stimulation of human lung fibroblasts with TGFβ. The cells of the invention also rescue small airway epithelial cells (SAECs) from cisplatin-induced apoptosis (Figure 21A) and stimulate greater than three-fold endothelial survival compared to the total monocyte population (Figure 21B). Normal endothelial regeneration and vascular remodeling are crucial for healthy repair and resolution of fibrosis following lung injury.

[0326] MSC-primed monocytes (i.e., the cells of the present invention) have potent anti-fibrotic function in vivo: delivery of the cells of the present invention to the hind limbs of mice following induction of ischemia results in a significant reduction in tissue fibrosis compared to delivery of monocytes cultured alone. Example 3

[0327] Clinical Safety Studies Five patients are injected with cells according to the invention.

[0328] Follow up the patient Pulmonary function CT scan 6-minute walk test QoL questionnaire Evaluation will be conducted. Example 4

[0329] Encapsulation In one embodiment, a population of cells of the invention is encapsulated. In this example, we demonstrate the encapsulation of monocytes together with MSCs.

[0330] Human monocytes and MSCs were prepared as a 3:1 mixture and encapsulated using alginate solution (1.5% w / v in 0.9% w / v NaCl) and CaCl2 according to our previously published technique (Ludwinski FE, Patel AS, Damodaran G, et al. Encapsulation of macrophages enhances their retention and angiogenic potential. NPJ Regen Med. 2019;4:6). Capsules were washed twice with Hank's Balanced Salt Solution (HBSS) through a 70 μM cell strainer (Corning, UK) and transferred to 6-well plates containing 2 ml of RPMI / 10% FCS medium for 3 days. After this time, cells were removed into a single cell suspension using trypsin / EDTA and analyzed for TIE2 and CD163 and CD206 expression via flow cytometry.

[0331] Encapsulation of monocytes with MSCs resulted in improved TIE2 (31%), CD206 (97.8%) and CD163 (58.7%) expression, suggesting a novel method to co-encapsulate cells for delivery rather than the need for co-culture (Figure 17).

[0332] Figure 17 shows alginate encapsulation of primary monocytes with MSCs. Monocytes exhibit high expression of TIE2, CD206 and CD163 after encapsulation without the need for adherent co-culture.

[0333] An advantage of the present invention is that the population of cells when encapsulated retains its triple positive characteristics, as demonstrated in FIG. Example 5

[0334] Production Monocytes are isolated from the blood of patients with CLI.

[0335] At steady state in patients, these monocytes are completely incapable of salvaging the ischemic limb in our preclinical mouse model.

[0336] These monocytes are co-cultured with MSCs.

[0337] MSCs can be obtained from any suitable source, in this example, MSCs are used from an in-house bank of adipose tissue-derived MSCs.

[0338] Co-culture is performed to drive / prime these monocytes, resulting in upregulation of their expression of three markers: TIE2, MRC1 and CD163. The combined presence of these markers then confers the cells of the invention the ability to stimulate new blood vessel growth to rescue ischemic limbs in mice.

[0339] A key step disclosed herein was optimizing the co-culture conditions by determining the optimal monocyte:MSC ratio and the length of time required for culture to stimulate a 20-fold increase in the number of Mo / MΦ expressing all three of these markers, ultimately comprising more than 80% of the total monocyte population.

[0340] Referring to FIG. 2, triple positive expressing monocytes show more than 20-fold enhancement when co-cultured with MSCs under our optimized conditions (B) compared to culture of monocytes without MSCs (A). Example 6

[0341] Optimized Production We optimized the conditions required to engineer triple-expressing TIE2+, MRC1+, CD163+ Mo / MΦ from total monocyte populations isolated from either CLI patients or controls.

[0342] The present inventors have tested the functional capacity of these modified cells and shown that the modified cells have the capacity to regulate blood vessel growth.

[0343] Functional in vitro assays show that conditioned medium from these MSC-primed Mo / MΦ enhances arteriogenesis (smooth muscle cell proliferation) compared to conditioned medium from monocytes cultured without MSCs (Figure 3). Conditioned medium is tested to demonstrate that the proteins produced by MSC-primed monocytes are functional.

[0344] FIG. 3 shows that MSC-primed Mo / MΦ stimulated smooth muscle cell proliferation to a greater extent than MO alone. * P<0.05, n=8; SMC=smooth muscle cells. Example 7

[0345] Revascularization using the cells of the present invention Our MSC-primed Mo / MΦ have a significantly higher ability to revascularize the ischemic limb in our mouse model of hindlimb ischemia HLI compared to non-primed control Mo / MΦ (FIG. 4).

[0346] FIG. 16A shows significantly greater revascularization of the ischemic hindlimb with MSC-primed MO compared to MO alone (P<0.001, * P<0.001. ** P<0.05 by post-hoc test, n=8 mice / group). Example 8

[0347] method Patient Recruitment Patients with CLI (Rutherford classification 4-6) who presented with leg tissue loss and / or rest pain, as well as age-matched controls (without clinical evidence of peripheral vascular disease) and healthy controls were recruited. Venous blood was collected in ethylenediaminetetraacetic acid (EDTA) tubes (BD Vacutainer, UK). Isolation of monocytes from whole blood is a well-established technique in our department. Briefly, PBMCs were first isolated from 100 mL of venous blood using Ficoll-Paque and magnetic immunobeads. Blood was first mixed 1:1 with RPMI-1640 and then layered on top of Ficoll-Paque reagent in a 2:1 (blood:Ficoll-Paque) ratio. Samples were centrifuged at 400 g for 30 min to remove mononuclear cells. Remaining red blood cells were lysed using BD Pharm Lyse (BD Biosciences). Following a washing step, the resulting cell suspension was blocked using FcR blocking reagent (Miltenyi Biotec) and incubated with anti-human CD14 microbeads, followed by immunomagnetic positive selection. Isolated monocytes were labeled with FITC-conjugated anti-human CD14 (BD Biosciences) and a dead cell staining dye (7-AAD; BD Biosciences) before analysis using flow cytometry (Attune, Thermo UK) to determine the purity and viability of the isolated cells.

[0348] primary cell culture Primary human adipose-derived mesenchymal stem cells (adMSCs) previously isolated, pooled and banked by our team in the Academic Department of Vascular Surgery at King's College London were used in this study. These adMSCs were maintained in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal calf serum (FCS, ThermoFisher Scientific), 1% antibiotic / antimycotic (ThermoFisher Scientific), 5ng / mL epidermal growth factor (EGF, R&D Systems), 1ng / mL basic fibroblast growth factor (bFGF, basic fibroblast growth factor), and 0.25ng / mL transforming growth factor (TGFβ, Millipore).

[0349] Isolated monocytes were cultured in monocyte medium (RPMI-1640 supplemented with 10% FCS and 1% antibiotic / antimycotic). All cells were cultured in a humidified incubator at 37°C and 5% CO2.

[0350] Co-culture technique of primary monocytes with adMSCs For co-culture experiments, frozen adMSCs were first thawed and seeded in these regular culture conditions for 2-3 days until the adMSCs were 80-90% confluent. Once confluency was achieved, the adMSC medium was removed and primary isolated monocytes were seeded in monocyte medium at a range of co-culture ratios. 10-15% more monocytes were added than the final number required, as experience from our laboratory dictates that approximately 10% of the cells will not adhere (see table below). The medium was changed on day 1 and then every 48 hours until harvest. [Table 2]

[0351] Collection of conditioned medium Monocytes in the co-cultures were magnetically re-separated from adMSCs using anti-CD45 beads (Miltenyi Biotec). The monocytes primed with adMSCs and those cultured alone were re-seeded into T25 flasks in RPMI containing 10% FCS. Conditioned medium was collected from the flasks after 48 hours of culture. The medium was centrifuged at 400g for 5 minutes to remove dead / floating cells. The supernatant was recentrifuged at 25000g for 10 minutes. The supernatant was flash frozen and stored at -80°C.

[0352] Flow cytometric analysis of cells, e.g., monocytes Extracellular phenotyping For flow cytometry analysis, cells were detached using trypsin-EDTA (Sigma Aldrich). The cell suspension was centrifuged at 400g for 5 min and then washed with wash buffer (PBS, 2 mM EDTA, 0.5% BSA). The cell suspension was blocked with human FcR blocking reagent for 20 min, after which a panel of fluorescently conjugated antibodies (Table 3) was added and cells were incubated for 30 min at 4°C in the dark and then washed. Cells were analyzed on an Attune™ NxT (ThermoFisher) flow cytometer using Attune™ NxT software v2.7.0. Compensation was performed using OneComp eBeads (ThermoFisher). For all samples, negative staining was determined using fluorescence minus-one (FMO) controls. The gating strategy consisted of exclusion of debris, doublets and dead cells, followed by CD45 +ve This involved gating the cells to identify monocytes. [Table 3]

[0353] Intracellular phenotyping Monocytes were centrifuged at 400g for 5 min, the supernatant was discarded, and cells were resuspended in 4% paraformaldehyde (PFA) for 20 min for fixation. Fixed samples were washed with wash buffer, centrifuged at 400g for 5 min, and the supernatant was discarded. Cell pellets were resuspended in 100 μL wash buffer and incubated with anti-human CD45 for 30 min at 4°C in the dark. Samples were washed with wash buffer, then centrifuged and resuspended in 100 μL perm / wash buffer (BD Biosciences) for cell permeabilization. Samples were stained with the intracellular panel for 30 min (Table 3). Samples were washed with perm / wash buffer and then resuspended for analysis. [Table 4]

[0354] Smooth muscle cell proliferation assay Human smooth muscle cells (SMC, Lonza, UK) were added at 1 × 10 3 Cells / well were seeded in 96-well plates in RPMI-1640 medium containing 10% FCS for 24 hours. Cells were then cultured overnight with conditioned medium (CM) for 24 hours. Control wells consisted of non-conditioned RPMI-1640 medium along with wells containing medium alone for background subtraction. After 24 hours, XTT (1 mg / ml) was added to each well and plates were cultured for 4 hours. Following this, absorbance was measured at 450 nm with a reference wavelength of 620 nm using a plate reader (Filter Max F5, Molecular Devices). Readings were taken every 2 hours until 12 hours, then at 22 hours and every 2 hours for a further 14 hours (total of 36 hours). Assays were performed in duplicate on 11 individual samples of primed / non-primed monocytes on days 3 and 7. All data were blinded and analyzed independently.

[0355] statistical analysis Data were analyzed using GraphPad Prism version 8 (GraphPad Inc.). Nonparametric tests were used for all experiments (Mann-Whitney U test for unpaired data, Wilcoxon paired signed-rank test for paired data). Data are presented as mean ± standard error of the mean (SEM) unless otherwise stated.

[0356] result Monocyte phenotype of adMSC-primed The phenotype of monocytes cultured alone or co-cultured with adMSCs was investigated over a series of time points (3, 7, 14 and 21 days) at a 2:1 monocyte:adMSC ratio using flow cytometry (n=6 samples per time point, see Figures 5 and 6 for examples of staining).

[0357] Day 3 Upregulated markers Monocytes co-cultured with adMSCs showed significantly increased positive expression of the "M2-like" receptors CD206, TIE2, and CD163 compared to monocytes cultured alone on day 3. In addition, co-culture with adMSCs also significantly increased expression of the "M1-like" receptor CD80.

[0358] Downregulated markers Co-culture of monocytes with adMSCs significantly reduced the expression of the "M2-like" receptor NRP1 compared with monocytes alone, and co-culture with ADMSCs significantly reduced the expression of HLA-DR.

[0359] Co-cultured monocytes showed no change in CD86 expression compared with monocytes cultured alone.

[0360] Day 7 Upregulated markers Monocytes co-cultured with adMSCs showed significantly increased positive expression rates of CD206, TIE2, and CD163, respectively, compared with monocytes cultured alone on day 7. Furthermore, co-culture with adMSCs significantly increased CD80 expression.

[0361] Co-culture of monocytes with adMSCs did not significantly alter the expression of CD86, NRP1, or HLA-DR compared to monocytes alone.

[0362] Day 14 Upregulated markers Monocytes co-cultured with adMSCs showed significantly higher positive expression rates of CD206, TIE2, and CD163 than those cultured alone by day 14. Co-culture with ADMSCs showed significantly higher expression of CD80. HLA-DR expression was also significantly higher.

[0363] Downregulated markers When monocytes were co-cultured with adMSCs, NRP1 expression was significantly decreased compared to monocytes alone. By day 14, co-culture with ADMSCs significantly decreased CD86 expression.

[0364] Day 21 Upregulated markers Monocytes co-cultured with adMSCs had significantly increased expression of CD206, TIE2, and CD163, respectively, compared with monocytes cultured alone on day 21. There was no difference in expression of HLA-DR and NRP1 when co-cultured with adMSCs. CD80 was also significantly increased.

[0365] When monocytes were co-cultured with adMSCs, there was no change in CD86 expression compared with monocytes alone.

[0366] Expression of proinflammatory CD38 is upregulated in MSC-primed monocytes We evaluated the key "M1-like" proinflammatory markers CD38 and CD80. The percentage of monocytic cells expressing CD38 is significantly increased after adMSC priming at both day 3 (62.2±7.1% vs. 91.7±2.6%, P<0.001, n=9, FIG. 7) and day 7 (54.7±6.4% vs. 85.0±4.4, P<0.01, n=8). CD80 expression was also significantly increased in co-cultures at day 3 of adMSC priming.

[0367] Figure 5 shows flow cytometric identification of monocytes. Monocytes in culture alone (A) or cultured with adMSCs (B) are identified according to CD45 expression after doublet and dead cell exclusion.

[0368] FIG. 6 shows examples of flow cytometry dot plots and histograms for the enhancement of CD206, TIE2, and CD163 and CD86 after co-culture of monocytes with adMSCs.

[0369] FIG. 7 shows improved CD38 expression after priming of monocytes with adMSCs. (A) Example flow cytometry dot plots of improved CD38 expression on day 3 after priming of monocytes with adMSCs. (B) Overall, CD38 expression is significantly improved after priming of monocytes with adMSCs. * P<0.05

[0370] [Table 5]

[0371] Optimization of cell ratios for monocyte priming with adMSCs The importance of monocyte:adMSC ratio for optimal monocyte priming was investigated (n=5 samples / ratio / time point, FIG. 8). TIE2 upregulation was greatest at a 3:1 ratio (7.6±3.3% vs. 84.2±12.2%). Enhancement of CD206 expression was higher at 2:1 and 3:1 ratios (12.2±2.7% vs. 92.3%±18.7% and 6.5±3.2% vs. 79±18.6%, respectively), while CD163 expression was highest at 5:1 (13.9%±5.4 vs. 88.8%±12.9%).

[0372] By day 7, expression of three "M2-like" markers cocultured with ADMSCs was significantly increased (but still lower than day 3) by a 5:1 ratio (TIE2: 13.46% ± 1.14 vs. 76.8% ± 14.18; CD206: 9.09% ± 0.89 vs. 74.4% ± 9.89; CD163: 13.45% ± 2.34 vs. 94.2% ± 18.7), representing a 4.2-, 11.0-, and 10.8-fold improvement, respectively (P < 0.0001 by Kruskal-Wallis test).

[0373] FIG. 8 shows a heat map illustrating the global optimization of fold-change increase in monocyte cell surface expression (compared to a 1:1 ratio for each time point).

[0374] Experimental validation of optimized monocyte priming The first set of experiments suggested that monocyte polarization was optimal on days 3-7 when co-cultured with adMSCs at a 3:1 ratio. Further experiments were performed (n=21 samples) to confirm and validate the time point that produced optimal monocyte phenotype. Monocyte polarization in co-culture was greater on day 3 compared to days 5 and 7 (CD206: 89.2% vs. 62.1% vs. 42.4%; TIE2: 75.3% vs. 50.4% vs. 32.3%; CD163: 78.3% vs. 72.4% vs. 28.9%, respectively). The fold-change increase in expression of each of the three markers was highest after 3 days of culture compared with days 5 and 7 (CD206: 13.3-fold vs. 4.1-fold vs. 2.8-fold; TIE2: 16.8-fold vs. 7.8-fold vs. 4.3-fold; CD163: 7.0-fold vs. 4.7-fold vs. 2.8-fold, respectively).

[0375] [Table 6]

[0376] FIG. 9 shows that 3 days after priming of monocytes with adMSCs, the increase in fold change was greatest in CD206, TIE2 and CD163.

[0377] Sequential flow cytometry gating confirms that day 3 is the optimal time point for the modification ("education" or "priming" or "production") of "triple positive" monocytes A gating strategy was used to determine the percentage of cells that were single, double and triple positive for the markers CD206, TIE2 and CD163 (Figure 10). Three days after priming with adMSCs, single receptor expression (CD206: 11.2±8.7% vs. 91.6±13.8%; CD163: 18.4±11.8% vs. 82.4±10.7%; TIE2: 9.1±7.1% vs. 82.6±9.1%) and dual receptor expression (CD206+ / CD163+: 4.9±4.1% vs. 80.7±13.6%; CD163+ / TIE2+: 5.8±5.3% vs. 73±9.5%; TIE2+ / CD206+: 5.4±4.5% vs. 78.1±12.9%) were significantly improved compared to monocytes cultured alone (P<0.0001 by Wilcoxon test, n=21 samples). Triple positive receptor expression of adMSC-primed monocytes (70.0±14.7%) was significantly higher than that of monocytes cultured alone (3.2±3.5%, P<0.0001, FIG. 11).

[0378] Seven days after adMSC priming, single receptor (CD206: 14.2±8.3% vs. 35.3±17.5%; CD163: 10.0±15.8% vs. 25.1±27.2%; TIE2: 11.5±11.9% vs. 31%±24.7) and dual receptor (CD206+ / CD163+: 0.5±0.5% vs. 22.9±17%; CD163+ / TIE2+: 0.4±0.5% vs. 24.8±22.2%; TIE2+ / CD206+: 1.5±2.8% vs. 22.2±16.2%) expression was improved in adMSC-primed monocytes compared to monocytes cultured alone (P<0.0001 by Wilcoxon test, n=21). Triple-positive receptor expression on adMSC-primed monocytes (16.3±15.3%) was significantly higher than that on monocytes cultured alone (0.09±0.1%, P<0.05, FIG. 11). However, there were much more triple-positive cells (70±14.7%) on day 3 of adMSC priming compared to day 7 (3.2±3.5%).

[0379] Figure 10 shows the sequential gating strategy for monocyte markers. CD45+ monocytes are gated on their single positive expression of CD206, CD163 and TIE2 based on FMO controls. Double positives are then identified by gating this population (R1) on the other two markers (R2 and R3). The R2 and R3 double positive populations are then gated on a third marker to determine the percentage of triple positive cells.

[0380] Figure 11 shows Venn diagrams of single, double and triple positive rates for CD206, CD163 and TIE2 expression in monocytes after 3 and 7 days of culture with and without MSCs. Single, double and triple positive staining is significantly higher after 3 days of ad-MSC priming compared to 7 days (n=21 samples, P<0.0001 by Wilcoxon test).

[0381] Proteomic profile of patient monocytes co-cultured with MSCs Monocytes co-cultured with adMSCs using our optimized technique do not display any known "M1" or "M2" phenotype. We determined the functional effects of co-culture by measuring proteins in the conditioned media of these cells. After 3 days of co-culture, HB-EGF and TGFβ were significantly decreased (both P<0.005, FIG. 12), and IL-12 was significantly higher (P<0.05). By day 7, there was no difference in TGFβ or IL-12 between monocytes co-cultured with adMSCs and those cultured alone, while HB-EGF was still significantly decreased (P<0.05).

[0382] Figure 12: Changes in the monocyte secretome following priming with adMSCs. Changes in IL12, TGFβ and HB-EGF are time-dependent.

[0383] Priming monocytes from patients with critical limb ischemia ("education") The cells / cell populations of the present invention can be used as autologous cell therapy. Therefore, we aimed to ensure that our optimized method can be used to prime monocytes isolated from patients with critical limb ischemia. Our optimized method resulted in significantly higher fold changes at day 3 compared to day 7 for TIE2 (16.1±5.3% vs. 2.2±0.4%, respectively, P<0.05), CD206 (7.2±2.6% vs. 1.4±0.1%, P<0.005) and CD163 (5.3±1.3% vs. 1.4±0.3%, P<0.05, FIG. 13).

[0384] Figure 13: Priming of monocytes from patients with CLI. After 3 days of priming, there is a significantly higher fold change in the expression of all three markers, TIE2, CD206 and CD163, compared to 7 days.

[0385] Similar to cultures of monocytes from control donors, triple positive receptor expression in monocytes from patients with CLI was significantly higher than in adMSC-primed monocytes after 3 days (adMSC-primed: 55.6 ± 10.3% vs. cultured alone: ​​0.3 ± 0.1%), but not after 7 days (0.3.6 ± 0.2% vs. 3.5 ± 3.4%, Figure 14 ).

[0386] Figure 14: Venn diagram of single, double and triple positive rates for CD206, CD163 and TIE2 expression in monocytes from CLI patients after 3 and 7 days of culture with or without MSCs. Single, double and triple positive staining is greater after 3 days but not 7 days of adMSC-priming (n=3 CLI samples).

[0387] In vitro and in vivo functions of adMSC-primed monocytes MSC-primed monocytes induce smooth muscle cell proliferation We compared our adMSC-primed monocytes with non-primed monocytes after 3 and 7 days of culture using our optimized ratio (3:1) for in vitro function. SMC proliferation was significantly improved using conditioned medium from monocytes primed with adMSCs for 3 days compared with both monocytes seeded alone and control medium (2-way repeated measures ANOVA, P<0.001, FIG. 15). On the other hand, there was no significant difference between primed and non-primed monocytes in vitro after 7 days of culture.

[0388] MSC-primed monocytes regulate postischemic neovascularization in vivo We compared our adMSC-primed monocytes with non-primed monocytes using our optimized ratio (3:1) and time point (3 days of priming) for in vivo function by delivering the cells into the hind limbs of ischemic mice.

[0389] Suitably, 1x10^6 cells are delivered. Suitably, said 1x10^6 cells comprise macrophage or monocyte cells, at least 50% of said macrophage or monocyte cells being characterized by expressing each of the markers: MRC1; TIE2; and CD163. Suitably, said cells are delivered in a saline buffer. Suitably, said cells are delivered by intramuscular injection. Suitably, a single dose of cells is administered.

[0390] We found that delivery of adMSC-primed monocytes significantly improved revascularization as seen by laser Doppler imaging of the ischemic limb compared to monocytes alone (P<0.01 by repeated measures two-way ANOVA, FIG. 16). Immunohistochemical analysis of injected muscles indicated that the mechanism for these improved revascularizations by adMSC-primed monocytes was the result of greater arteriogenesis in the target tissue via an increase in the number and diameter of arterioles.

[0391] Figure 15: Smooth muscle cell proliferation potential of adMSC-primed monocytes and monocyte cultures alone. Graph example of XTT measurements showing greater smooth muscle cell (SMC) proliferation after culture with conditioned medium (CM) from adMSC-primed monocytes compared to non-primed monocytes after 3 days (A), but not after 7 days of priming (B). (c) Overall, from day 3 onwards, there is a significant improvement in SMC proliferation in response to CM from adMSC-primed monocytes compared to CM from monocytes cultured alone. This is not seen after 7 days of culture, where there is no difference in SMC proliferation from adMSC-primed monocytes compared to monocytes alone (D) (n=11 separate monocyte samples, each performed in duplicate).

[0392] Figure 16 shows revascularization of adMSC-primed monocytes and monocyte cultures alone delivered to the ischemic hindlimb. (A) Delivery of adMSC-primed monocytes to the ischemic hindlimb results in significantly greater revascularization compared to monocytes cultured alone by days 21 and 28 (P<0.01 by 2-way repeated measures ANOVA, *P<0.05 by post-hoc Bonferroni test; ** *P<0.01, n=5 mice / group). (B) Example laser Doppler images of mice treated with monocytes cultured alone compared to adMSC-primed monocytes, showing greater perfusion of the paw by days 21 and 28. (C) Example immunohistochemistry showing greater size of arterioles (SMA staining, red) in muscle from mice following treatment with adMSC-primed monocytes compared to monocytes cultured alone. (D) Overall, treatment of limbs with adMSC-primed monocytes significantly increases arteriogenesis (number of arterioles) compared to monocytes cultured alone (n=9 mice / group).

[0393] Figure 17 shows the phenotype of monocytes after co-encapsulation with MSCs. These monocytes upregulate their expression of TIE2, MRC1 and CD163.

[0394] FIG. 18 shows that MSC-primed monocytes exhibit significantly downregulated expression of MMP-9 compared to monocytes cultured alone.

[0395] Figure 19 shows that MSC-primed monocytes express significantly higher levels of HGF, IL-10 and TNF-α. These proteins are known to have significant pro-angiogenic / pro-arteriogenic and anti-fibrotic activities. Such cells can also further upregulate the expression of IL-10 and TNF-α when exposed to inflammatory stimuli such as lipopolysaccharide (LPS). Furthermore, the primed state of such cells is not restored by exposure to LPS, as their expression of HGF is not reduced.

[0396] Figure 21 shows picrosirius red staining of mouse adductor muscles after delivery of monocytes cultured alone (top) and MSC-primed monocytes. The product prevents tissue damage and fibrosis after ischemia. Representative images taken using polarized light microscopy from eight mice in each group. White arrows indicate areas of collagen deposition (green and orange / yellow fibers) that are reduced in mice treated with MSC-primed monocytes. Overall, there is a significant reduction in fibrosis with treatment with MSC-primed monocytes compared to the total monocyte population (mean 58% ± 4 SEM; *P < 0.005). Example 9

[0397] Safety Profile Here, we present safety evaluation data in mammals (mice).

[0398] In this experiment, six mice were inoculated with 1 × 10 6 MSC-primed monocytes (cells of the invention) were injected intravenously via the tail vein into nude athymic mice (n=6).

[0399] Survival rate was 100% at 6 weeks.

[0400] We refer to FIG.

[0401] This demonstrates the expected safety of the cells of the invention for human use. Example 10

[0402] Clinical Cell Manufacturing The initial donor product (starting cells) can be either whole blood or leukapheresis product. In the case of whole blood, red blood cell volume reduction using the "WB Step 1" cell washing program on a Lovo™Med device (Fresenius Kabi, Three Corporate Drive Lake Zurich, IL 60047, USA) is first required, followed by monocyte labeling, whereas in the case of leukapheresis product, this is not necessary. Monocytes / macrophages are then separated by labeling the cell suspension with anti-CD14 magnetic beads and passing it through a magnetic column CliniMACS Plus cell processor (catalog number 151-01 Miltenyi Biotec, address ibid.) to enrich for CD14+ cells.

[0403] "Priming" (co-culture with MSCs) The first step is to generate a bank of MSC vials that can then be thawed for the production of each clinical batch. MSCs are sourced from RoosterBio Inc, 5295 Westview Drive, Suite 275, Frederick, MD 21703 (RoosterVial™-hBM-20M-XF, MSC-CC040) and are produced and expanded according to the manufacturer's guidelines before storage (10 7 Pieces or 10 8 Both formats are available in various quantities depending on the scale of production, resulting in a co-culture in vials containing either 1000 cells.

[0404] The next step is to culture these monocyte / macrophage cells with a confluent layer of MSCs, usually for a period of up to 7 days, more suitably for a period of up to 5 days.

[0405] To generate this confluent layer, 10 cells were cultured per CellSTACK, Corning Cat. No. 3330. 7 Therefore, if CellSTACK 10 (Corning Cat. No. 3312) is used for large-scale production, 10 8 1 vial of MSCs must be thawed. The MSCs are thawed 72 hours prior to co-culture with monocytes / macrophages and cultured in an appropriately sized CellSTACK with Rooster Nourish medium for 72 hours. This results in a confluent (>80%) layer of MSCs within the CellSTACK. After 72 hours, monocytes / macrophages are added to the MSCs, at which point the medium is changed to X-vivo 10 medium (Lonza).

[0406] No medium change is necessary, however, if TIE2 levels have not improved after 5 days of culture, the culture period may be extended to 7 days (including a medium change on day 5).

[0407] By "enhanced" herein is meant expression that is increased to a level higher than in the starting cell.

[0408] Suitably, "enhanced" means expression in at least 50% of the monocytes / macrophages present to a level higher than in the starting cells.

[0409] The co-cultured cells are then detached, re-incubated with anti-CD14 beads, and processed again through the magnet to separate the monocytes / macrophages from the MSCs. The resulting monocyte-enriched population of cells can be analyzed for purity via CD45 expression, as MSCs do not express this marker.

[0410] The cells may then be formulated in Plasma-Lyte 148 supplemented with 5% v / v human serum albumin and 10% v / v DMSO and frozen in a controlled rate freezer. Example 11

[0411] Exemplary Methods of Manufacturing Step 1 The initial donor product can be either whole blood or a leukapheresis product. For whole blood, 350-485 mL of blood is collected and the volume reduced to approximately 150 mL using the "WB Step 1" cell wash program on a Lovo™ Med device (Fresenius Kabi). The resulting red blood cell reduced suspension is collected into a transfer bag. For leukapheresis products, the cell suspension does not require volume reduction.

[0412] Step 2: Enrichment of CD14+ monocytes If the initial donor product is whole blood, transfer 1 / 100th of the erythrocyte reduction volume (approximately 1.4-1.5 ml) of CliniMACS CD14 Reagent (Miltenyti Biotec, 170-076-705) to the volume-reduced cell suspension. If the initial product is leukapheresis, transfer 5 mL of CD14 Reagent. Incubate the reagent at 2-10°C for 20 min with continuous gentle agitation. Following incubation, process the cells through a CliniMACS Plus cell processor (Miltenyi Biotec) using the pre-set program for enrichment (Enrichment 3.2) according to the manufacturer's written instructions. Collect CD14+ target cells in a cell collection tube. Upon completion of the automated program, centrifuge the collection tube containing the CD14+ enriched monocytes at 750 x g for 10 min. The cells are then resuspended in 40 mL of X-Vivo 10 medium (Lonza).

[0413] Step 3: Co-culture For first donors of whole blood: total monocyte count 1 x 10 8If the number of cells is less than 1 × 10, seed the cells onto one CellSTACK (Corning, 3330). 8 When more than 100 monocytes are isolated from the patient's blood, the monocytes are then seeded onto two CellSTACKs (Corning 3310).

[0414] For leukapheresis donors: up to 1 × 10 9 Monocytes are seeded onto CellSTACK-10 (Corning, 3312). Cells are cultured on a confluent (80-90% confluence) layer of MSCs (RoosterBio) for a period of typically up to 5 days. No medium change is required.

[0415] If the percentage of monocytes expressing TIE2 is below 50% after 5 days of culture, the culture period may be extended to 7 days (including a medium change on day 5).

[0416] Step 4: Product Harvesting To detach adherent cells from the CellSTACK, remove the medium and add 20 mL pre-warmed TrypLE (ThermoFisher, A1285901) to each CellSTACK layer for 12 min in a 37 °C incubator with agitation every 3-4 min. Quench the TrypeLE with CliniMACS PBS / EDTA buffer + 0.5% HAS. Bring the final volume to 180 ml (using Lovo for volume reduction as above if the original donor product was leukapheresis). Re-incubate the monocyte-MSC suspension with 1.8 ml CliniMACS CD14 Reagent (1 / 100). Monocytes can then be separated from MSCs using either the CliniMACS Plus Enhanced Program or a magnetic separation LS column and a QuadroMACS Separator (Miltenyi Biotec). An LS column is necessary if the initial product is whole blood, since the cell count is lower and in this situation CliniMACS Plus is not sufficient to ensure high yield and purity. The retained cells are resuspended in a total volume of 55 mL of PlasmaLyte-148 + 10% HSA. The resulting monocyte-enriched population of cells can be analyzed for purity by determining the percentage of cells expressing CD45 expression, since MSCs do not express that marker. The cells are formulated in Plasma-Lyte 148 supplemented with 5% v / v human serum albumin and 10% v / v DMSO and frozen in a controlled rate freezer. Example 12

[0417] Manufacturing repeatability We present data showing the reproducibility of the method (manufacturing).

[0418] We refer to Figure 4, which shows bar graphs demonstrating reproducibility data for CD206, CD163 and TIE2 expression by three separate technicians generating the cells of the invention (MSC-primed monocytes) as in the above example: Technician A: n=10 samples, B: n=6 samples, C: n=5 samples.

[0419] No significant differences were found between technicians for all three markers, therefore the disclosed method is reproducible. Example 13

[0420] 25F9 We refer to FIG.

[0421] There is higher expression of 25F9 in MSC-primed monocytes at all time points compared to monocytes cultured alone. Monocytes cultured alone do not increase their expression of 25F9 until day 7, whereas MSC-primed monocytes increase their expression levels depending on the culture conditions.

[0422] We refer to FIG.

[0423] Flow cytometry dot plot showing sequential gating of circulating monocytes in blood. In this example, 10,365 monocytes were analyzed in this blood sample, of which 8 / 10,365 cells are triple positive (0.08%).

[0424] Blood monocytes (primary monocytes) do not express the marker 25F9, whereas MSC-primed cells (cells of the invention) significantly upregulate this marker by day 3. Example 14

[0425] Sustained expression / effective timing of induction The prior art expression levels of various markers are significantly lower than in the cells of the present invention and are not clinically useful.

[0426] The inventors also demonstrate that these levels drop further with increasing culture time, highlighting the valuable contribution of timing in the methods disclosed herein.

[0427] It should be noted that in prior art methods, monocytes are first cultured alone for several days (resulting in differentiation into macrophages) before being co-cultured with MSCs, resulting in most or all cells being cultured for more than 7 days. In contrast, the present invention teaches direct co-culture of monocytes (primary monocytes) with MSCs from day 0.

[0428] We refer to Figures 28, 29 and 30 which show this, namely that the levels of three key markers CD202B (TIE2), CD163 and CD206 (MRC1) drop after day 7, i.e., levels are prominent within the timings taught herein and levels drop when cultured outside the timings taught herein. Example 15

[0429] Human evidence The MONACO Cell Therapy Study: Monocytes as Antifibrotic Treatment Post-COVID-19 (NCT0480508) is a Phase 1, open-label trial evaluating the safety and tolerability of a single intravenous dose of our product (i.e., cells of the invention) (i.e., MSC-primed monocytes) in patients with fibrotic lung disease following COVID-19 infection. Five patients were recruited into the study.

[0430] We found that the median absolute FVC was significantly improved at weeks 12 and 24, respectively, compared to baseline (Figure 31(i), *P<0.05). We also observed a significant improvement in walking distance at week 24 (Figure 31(ii), *P<0.05), and improvements in shortness of breath and K-BILD scores (Figures 31(v) and 31(vi)).

[0431] Figure 31(iii) and Figure 31(iv) show a patient with improvement in areas of pulmonary fibrosis (see arrow) (red arrow in file) following infusion of MSC-primed monocytes.

[0432] Thus, the beneficial technical effects of the present invention have been demonstrated in human subjects.

Claims

1. A population of cells, comprising at least 50% myeloid cells, e.g., macrophages or monocytes; At least 50% of the bone marrow cells, e.g., macrophages or monocyte cells, express the marker: - MRC1; TIE2; and ・CD163 A population of the above cells, characterized in that each of the above expresses each of the above.

2. 2. The population of cells of claim 1, comprising at least 70% myeloid cells, such as macrophages or monocytes.

3. 2. The population of cells of claim 1, comprising at least 80% myeloid cells, such as macrophages or monocytes.

4. At least 60% of bone marrow cells, e.g., macrophages or monocytes, express the marker: - MRC1; TIE2; and ・CD163 10. The population of cells of claim 1, wherein each of the cells expresses:

5. The population of cells of claim 1 , wherein the myeloid cells, such as macrophages or monocyte cells, express CD14 and / or CD45.

6. 2. The population of cells of claim 1, wherein the myeloid cells, such as macrophages or monocyte cells, express TNF alpha (TNFa) and / or IL-12.

7. 2. The population of cells of claim 1, comprising a 3:1 ratio of bone marrow cells, e.g., monocyte / macrophage cells:MSCs.

8. (a) providing monocytes from a subject (b) providing an MSC; (c) culturing the monocytes with the MSCs A method comprising: The method, wherein step (c) comprises contacting the monocytes with the MSCs to produce a cell mixture, and culturing the cell mixture.

9. 9. The method of claim 8, wherein in step (c) the ratio of monocytes:MSCs is 3:

1.

10. The method according to claim 8 or 9, wherein the cells are cultured for about 3 to 7 days, preferably about 3 to 5 days.

11. 11. The method of claim 10, wherein the cells are cultured for about 3 days.

12. 10. The method of claim 8 or 9, wherein the cells are cultured in a medium and the medium is changed every 5 days.

13. A therapeutic agent for peripheral vascular disease, suitably comprehensive severe chronic lower limb ischemia, comprising a population of cells according to any one of claims 1 to 7.

14. A therapeutic agent for fibrosis, comprising a population of cells described in any one of claims 1 to 7.

15. A therapeutic agent for ischemic stroke, comprising a population of cells described in any one of claims 1 to 7.

16. A therapeutic agent for a mammalian subject, comprising the population of cells according to any one of claims 1 to 7.

17. The method of claim 16, wherein a dose of 100 million to 200 million cells is administered.

18. An inducer of angiogenesis in a mammal, comprising a population of cells described in any one of claims 1 to 7.

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