Reprogrammed smooth muscle cells and methods related thereto

Abstract

Provided herein are novel reprogrammed smooth muscle cells (rSMC) and methods of making and using the cells to treat ischemia. The rSMC is produced by culturing a fibroblast with all trans-retinoic acid (ATRA) under conditions to produce the rSMC from the fibroblast, wherein the fibroblast is genetically modified to overexpress cardiocardin. The rSMC is more advantageous than currently available regenerative vascular therapies by promoting vascular perfusion in a recipient subject. In particular, the rSMC may increase neovascularization of small and large vessels.

Description

[0001] PRIOR RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 428,579, filed on November 29, 2022, which is hereby incorporated by reference in its entirety.

[0003] STATEMENT REGARDING FEDERAL GOVERNMENT SPONSORED RESEARCH

[0004] This invention was made with government support under Grant Nos. HL150887 and HL157242 awarded by the National Institutes of Health. The government has certain rights in the invention. REFERENCE TO A SEQUENCE LISTING SUBMITTED IN XML FORMAT VIA THE PATENT CENTER

[0005] This application contains a Sequence Listing in XML format. The Sequence Listing, named 043150 - 1413091.xml, was created on November 29, 2023, is 97 kilobytes in size, and is hereby incorporated by reference in its entirety. BACKGROUND OF THE INVENTION

[0006] Ischemic cardiovascular disease is a leading cause of morbidity and mortality worldwide. Vascular insufficiency is a hallmark pathological feature of ischemic cardiovascular disease. Regenerative vascular therapies have been developed to promote new blood vessel formation. However, such regenerative therapies are typically limited to blood vessels the size of capillaries. Additionally, regenerative therapies lack the ability to promote the recruitment of mural cells (i.e., pericytes and vascular smooth muscle cells (SMCs)) to small or large blood vessels. Thus, currently available therapies lack the ability to effectively perfuse ischemic tissues and organs. SUMMARY OF THE INVENTION

[0007] Provided herein is a method for generating reprogrammed smooth muscle cells (rSMCs) by culturing fibroblasts with all - trans retinoic acid (ATRA) under conditions for generating rSMCs from fibroblasts, wherein the fibroblasts are genetically modified to express or over - express myocardin. The conditions for generating rSMCs from fibroblasts include contacting the fibroblasts with ATRA for at least two days (including, for example, 4 - 8 days). The fibroblasts are optionally mammalian fibroblasts, or more specifically human fibroblasts. For example, the genetically modified fibroblasts are optionally human dermal fibroblasts.

[0008] The fibroblasts can be genetically modified by any method known in the art to introduce a heterologous nucleic acid encoding myocardin into the fibroblasts. Such methods include, but are not limited to, viral transduction. Optionally, the heterologous nucleic acid is stably integrated into the fibroblast genome, for example, by gene editing.

[0009] The present disclosure also provides rSMCs or progeny thereof that contain heterologous nucleic acids. The rSMCs contain heterologous nucleic acids encoding myocardin, and the rSMCs co-express CNN1 and SMTN in a non-striated pattern. Optionally, the rSMCs are prepared by any of the methods described herein. In the presence of carbachol or another agent that promotes intracellular calcium release, the rSMCs contract by more than 10%.

[0010] Also provided is a composition that includes a population of rSMCs and a pharmaceutically acceptable carrier. Such a composition is designed for administration to a subject.

[0011] Also provided is a method of treating a subject having ischemia or at risk of developing ischemia, the method including administering to the subject an effective amount of rSMCs or a composition that includes a population of rSMCs and a carrier. The effective amount of rSMCs or composition increases the vascular perfusion of the subject, increases the neovascularization of the subject, and / or increases the arteriogenesis of the subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] This application includes the following drawings. The drawings are intended to illustrate certain embodiments and / or features of the compositions and methods, and are intended to supplement any one or more descriptions of the compositions and methods. The drawings do not limit the scope of the compositions and methods, unless the written description clearly indicates otherwise.

[0013] Figure 1 is an exemplary schematic diagram of an experimental design in which human dermal fibroblasts (HDFs) were treated with all-trans retinoic acid (ATRA) for four days, showing an increase in ATRA concentration over the four-day period.

[0014] Figure 2 Shows an increase in STRA6 expression in ATRA-treated HDFs compared to untreated HDFs, and the increase is more pronounced at higher ATRA concentrations.

[0015] Figures 3A - 3H Shows an increase in the expression of eight genes specific to the contractile SMC phenotype ( Figure 3A , MYOCD; Figure 3B , ACTA2; Figure 3C , TAGLN; Figure 3D , CALD1; Figure 3E , DES; Figure 3F , CNN1; Figure 3G , MYH11; and Figure 3H , SMTN) in ATRA-treated HDFs compared to untreated HDFs. A concentration of 2 μM ATRA was the most effective concentration tested for induction of SMC gene expression.

[0016] Figure 4It shows that compared with untreated HDFs, the expression of S100A4 in ATRA-treated HDFs is decreased.

[0017] Figure 5 Is an exemplary schematic diagram of the experimental design for generating MYOCD-transduced HDFs with ATRA treatment. HDFs were transduced with MYOCD on day 0 and day 2, and then cultured with ATRA until day 16.

[0018] Figures 6A - 6H It shows that compared with untransduced HDFs or MYOCD-treated HDFs not treated with ATRA as Figure 5 shown, in HDFs transduced with MYOCD and treated with ATRA (MATRA-treated HDFs), the expression of eight genes ([ Figure 6A , MYOCD; Figure 6B , ACTA2; Figure 6C , TAGLN; Figure 6D , CALD1; Figure 6E , DES; Figure 6F , CNN1; Figure 6G , MYH11; and Figure 6H , SMTN) specific to the contractile SMC phenotype is increased. The gene expression of all genes reaches a peak at day 4, except that the expression of SMTN reaches a peak at day 8.

[0019] Figure 7 Is an exemplary schematic diagram of the experimental design for generating reprogrammed SMCs from HDFs. HDFs were transduced with MYOCD on day 0 and day 2, and then cultured with ATRA until day 4.

[0020] Figures 8A - 8H It shows that compared with untransduced HDFs and MYOCD-HDFs, at day 4, in MATRA-treated HDFs as Figure 7 shown, the gene expression of eight genes ([ Figure 8A , MYOCD; Figure 8B , ACTA2; Figure 8C , TAGLN; Figure 8D , CALD1; Figure 8E , DES; Figure 8F , CNN1; Figure 8G , MYH11; and Figure 8H , SMTN) specific to the contractile SMC phenotype is significantly increased.

[0021] Figure 9 It shows that compared with untransduced HDFs and MYOCD-HDFs not treated with ATRA, at day 4, in MATRA-treated HDFs, the gene expression of STRA6 is significantly increased.

[0022] Figures 10A - 10C shows that, compared with untransduced HDFs and MYOCD-HDFs not treated with ATRA, on day 4, in MATRA-treated HDFs, the gene expression of Meta-VCL ( Figure 10A ), TPM1 ( Figure 10B ), and TPM2 ( Figure 10C ), which regulate SMC contraction, was significantly increased.

[0023] Figures 11A - 11D shows that, compared with untransduced HDFs and MYOCD-HDFs not treated with ATRA, on day 4, in MATRA-treated HDFs, the gene expression of four genes (KLF4 ( Figure 11A ), MYH10 ( Figure 11B ), MMP2 ( Figure 11C ), and SPP1 ( Figure 11D )) specific for synthetic SMCs was significantly decreased.

[0024] Figure 12 shows that, compared with untransduced HDFs, on day 4, in MATRA-treated HDFs, the gene expression of CSPG4 (pericyte gene) was increased. The CSPG4 gene expression in MYOCD-HDFs and MATRA-treated HDFs was higher than that in untransduced HDFs.

[0025] Figures 13A - 13D shows that, compared with untransduced HDFs and MYOCD-HDFs, on day 4, in MATRA-treated HDFs, the gene expression of S100A4 ( Figure 13A ), THY1 ( Figure 13B ), VIM ( Figure 13C ), and PDGFRA ( Figure 13D ), which represent fibroblasts, was decreased.

[0026] Figure 14 shows the flow cytometry analysis of ACTA2 expression in HDFs, MYOCD-HDFs, and MATRA-treated HDFs. On day 4, compared with approximately 5.0% and 35.3% of cells expressing ACTA2 in the treatment groups of untransduced HDFs and MYOCD-HDFs without ATRA, respectively, approximately 57% of MATRA-treated HDFs expressed ACTA2.

[0027] Figure 15Flow cytometry analysis of MYH11 expression in HDF, MYOCD-HDF, and MATRA-treated HDF is shown. At day 4, approximately 48% of the cells in MATRA-treated HDF expressed MYH11, compared to approximately 3.6% and 10.2% of the cells expressing MYH11 in the untransduced HDF and MYOCD-HDF groups, respectively.

[0028] Figure 16 Flow cytometry gating strategy for selecting single live cells in MATRA-treated HDF, MYOCD-HDF, and untransduced HDF groups is shown.

[0029] Figure 17 Flow cytometry gating strategy for identifying ACTA2+ cells in MATRA-treated HDF, MYOCD-HDF, and untransduced HDF groups is shown.

[0030] Figure 18 Flow cytometry gating strategy for identifying MYH11+ cells in MATRA-treated HDF, MYOCD-HDF, and untransduced HDF groups is shown.

[0031] Figure 19 Micrographs of untransduced HDF, MYCOD-HDF, and MATRA-treated HDF are shown, with double immunofluorescence staining for the contractile SMC markers ACTA2 (first column) and TAGLN (second column), and DAPI (middle column) used as a nuclear counterstain. The merged staining of TAGLN, DAPI, and ACTA2 is shown in the two right columns. The boxed area in the fourth column is shown magnified further in the fifth column. MATRA-treated HDF robustly co-expresses ACTA2 and TAGLN compared to MYOCD-HDF and untransduced HDF.

[0032] Figure 20 Micrographs of untransduced HDF, MYCOD-HDF, and MATRA-treated HDF are shown, with double immunofluorescence staining for CNN1 (first column) and SMTN (second column), and DAPI (third column) used as a nuclear counterstain. The merged staining of all three is shown in the two right columns. In the rightmost column, the boxed area is shown magnified further. MATRA-treated HDF robustly expresses CNN1 and SMTN compared to MYOCD-HDF and untransduced HDF.

[0033] Figure 21Confocal microscopy images (left) of untransduced HDF, MYOCD-HDF, MATRA-treated HDF, and human aortic SMC (HAoSMC) are shown, as well as a graph (right) showing the change in cell surface area before and after carbachol treatment (the dotted line in the bottom row indicates the contracted state, and the solid line indicates the non-contracted state). In the presence of carbachol, MATRA-treated HDF contracted by approximately 28% as measured by cell surface area. HAoSMC contracted by approximately 32%, while untransduced HDF contracted by approximately 9%, and MYOCD-HDF contracted by approximately 20%.

[0034] Figure 22 Photographs (left) of collagen matrices carrying untransduced HDF, MYOCD-HDF, MATRA-treated HDF, and HAoSMC are shown, as well as a graph (right) showing the change in cell surface area two days after treatment with carbachol (dotted line). In the presence of carbachol, MYOCD-HDF and MATRA-treated HDF showed stronger contraction than HAoSMC and untransduced HDF in the collagen gel.

[0035] Figure 23 Confocal microscopy images of Fluo-4 (GFP)-preloaded untransduced HDF, MYOCD-HDF, MATRA-treated HDF, and HAoSMC before and after treatment with carbachol are shown. In the presence of carbachol, the amount of intracellular calcium released by MATRA-treated HDF was similar to that of HAoSMC, as shown by the fluorescence intensity in the bottom panels of each group. MATRA-treated HDF and HAoSMC both showed more intracellular calcium release compared to untransduced HDF and MYOCD-HDF.

[0036] Figure 24 A graph showing the percentage of Fluo-4-preloaded cells responsive to carbachol is shown. After carbachol treatment, MATRA-treated HDF had a significantly higher rate of intracellular calcium release response compared to HAoSMC, MYOCD-HDF, and untransduced HDF.

[0037] Figure 25 Is an exemplary schematic diagram of parameters (main peak, time to reach maximum fluorescence (F / F0), F / F0max, and multiple peaks) for evaluating intracellular calcium release.

[0038] Figure 26 Shows the maximum fluorescence intensity (F / F0 max), indicating the amount of intracellular calcium release after carbachol treatment as shown in Figure 25 The calcium release in MATRA-treated HDF was significantly higher compared to HAoSMC, MYOCD-HDF, and untransduced HDF.

[0039] Figure 27 shows the time to reach F / F0 max, the fluorescence indication such as Figure 25 the time to reach maximum intracellular calcium release as shown. The time to reach F / F0 max was significantly shorter for MATRA-treated HDFs, MYOCD-HDFs, and HAoSMCs compared to untransduced HDFs.

[0040] Figure 28 shows an F / F0 overlay plot within 550 seconds of five randomly selected untransduced, carbachol-treated HDFs. In response to carbachol treatment, untransduced HDFs produced one or no major peaks of intracellular calcium release.

[0041] Figure 29 shows an F / F0 overlay plot within 550 seconds of five randomly selected carbachol-treated MYOCD-HDFs. In response to carbachol treatment, MYOCD-HDFs produced a large major peak of intracellular calcium release, followed by a small number of repetitive peaks.

[0042] Figure 30 shows an F / F0 overlay plot within 550 seconds of five randomly selected MATRA-treated HDFs (labeled rSMCs) in response to carbachol treatment. In response to carbachol treatment, MATRA-treated HDFs produced a more robust major peak of intracellular calcium release, followed by repetitive peaks over a longer period.

[0043] Figure 31 shows an F / F0 overlay plot within 550 seconds of five randomly selected HAoSMCs in response to carbachol treatment. In response to carbachol treatment, HAoSMCs produced a large major peak of intracellular calcium release, followed by a small number of repetitive peaks.

[0044] Figure 32 shows F / F0 overlay plots of calcium release in response to carbachol treatment for a single untransduced HDF, MYOCD-HDF, MATRA-treated HDF, and HAoSMC. In response to carbachol treatment, MATRA-treated HDFs produced a robust major peak of intracellular calcium release, followed by a propagating wave of calcium release events during the test period. However, MYOCD-HDFs and HAoSMCs each produced a large major peak, followed by a small number of repetitive peaks, while untransduced HDFs did not produce a major peak.

[0045] Figure 33 shows phase contrast micrographs before and 24 hours after performing a scratch wound healing assay and a graph showing the percentage of wound healing in each treatment group. Significantly fewer MATRA-treated HDFs and HAoSMCs migrated into the scratch wound (indicated by the dashed line in the figure) compared to untransduced HDFs and MYOCD-HDFs.

[0046] Figure 34 Transmission electron micrograph images of untransduced HDF, MYOCD-HDF, and MATRA-treated HDF are shown. The boxed regions in the left column are further magnified in the right column, and certain cellular components are labeled. Compared with MYOCD-HDF and untransduced HDF, MATRA-treated HDF has more contractile filaments anchored by dense bodies (DB), but significantly fewer mitochondria (M) and free ribosomes.

[0047] Figure 35 Results of principal component analysis of four different gene expression groups in MATRA-treated HDF (designated as rSMC), HAoSMC, MYOCD-HDF, and untransduced HDF, evaluated using RNA sequencing data, are shown.

[0048] Figure 36 A clustering heatmap is shown, depicting the correlations between HDF, MYOCD-HDF, MATRA-treated rSMC, and HAoSMC by the Pearson correlation coefficient method. The heatmap shows four different gene expression groups divided by cell type. Low correlation coefficients are shown in dark gray, while high correlation coefficient values are shown in light gray.

[0049] Figure 37 A variance plot of principal component analysis of RNA sequencing data is shown. MATRA-treated cells (rSMC) show a different gene expression pattern from HAoSMC, MYOCD-HDF, and untransduced HDF, and there is little difference between replicates.

[0050] Figure 38 A clustering heatmap of gene expression in cell types is shown. Low correlation coefficients are shown in dark gray, while high correlation coefficient values are shown in light gray. Evaluated by principal component analysis of RNA sequencing data, rSMC shows a gene expression pattern similar to HAoSMC, but different from the gene expression patterns in MYOCD-HDF and untransduced HDF.

[0051] Figure 39 A Venn diagram is shown, presenting the number and overlap of differentially expressed genes (DEG) between groups. DEG are sorted by relative expression to untransduced HDF. Compared with MYOCD-HDF, rSMC shows a high overlap of differentially expressed genes with HAoSMC.

[0052] Figures 40A - 40D An Mfuzz cluster is shown, presenting the upregulated gene expression patterns for each group ( Figure 40A , HDF; Figure 40B , MYOCD-HDF; Figure 40C , rSMC;Figure 40D , HaoSMC). A subset of the most significantly regulated genes in each group is shown. Gene Ontology (GO) terms are shown. BP is biological process, CO is cellular component, HP is human phenotype, and MF is molecular function. Genes highly expressed in HDF are related to mitosis and cell proliferation (KIF4A, CDCA8, CENPA, and GTSE1); genes highly expressed in MYOCD-HDF include endolysosomal genes (LYPLA2, TPP1, and TANGO2) and cytoskeletal genes (EPS8L2 and SYNE3); genes highly expressed in rSMC include muscle development and function genes (SORBS1, CNN1, and MYL7) and embryonic skeletal system morphogenesis genes (HOXD13 and HOXC13); and genes highly expressed in HaoSMC include signal transduction system genes (MRAP2, SULT1E1, CACNG8, and CD200).

[0053] Figures 41A - 41D The Mfuzz clusters are shown, demonstrating the downregulated gene expression patterns for each group ( Figure 41A , HDF; Figure 41B , MYOCD-HDF; Figure 41C , rSMC; Figure 41D , HaoSMC). The GO terms are as described in Figure 40. The downregulated gene expression in MYOCD-HDF is related to biological processes; the downregulated gene expression in rSMC of genes is related to cellular components; and the downregulated gene expression in HaoSMC of genes is related to human phenotypes. The number of downregulated genes in each group ranges from 632 to 1,013.

[0054] Figures 42A - 42J A graph showing the expression levels of ten genes specific to the SMC phenotype (shown as Log2 counts per million mapped reads (Log2 CPM)) is shown ( Figure 42A , ACTA2; Figure 42B , CALD1; Figure 42C , CNN1; Figure 42D , MYH11; Figure 42E , MYL6; Figure 42F , MYOCD; Figure 42G , TAGLN; Figure 42H , TPM1; Figure 42I , TPM2; and Figure 42J , VCL). The expression in MATRA-treated HDF (rSMC) is significantly higher than that in HaoSMC, MYOCD-HDF, and untransduced HDF.

[0055] Figure 43A volcano plot is shown, demonstrating differential gene expression of untransduced HDFs compared to rSMCs.

[0056] Figure 44 A dendrogram showing GO terms enriched in rSMCs is shown. Genes were sorted by relative expression compared to HDF expression. Compared to gene expression in untransduced HDFs, rSMCs are enriched in the expression of genes related to contraction and muscle development.

[0057] Figure 45 A gene network diagram of tissue morphogenesis, skeletal system development, striated muscle tissue development, muscle tissue development, and cardiac muscle tissue development divided by gene ontology terms is shown. The dot size for each term (as Figure 46 shown) indicates the number of genes assigned to that term, with the smallest dot representing 20 genes and the largest dot representing 60 genes.

[0058] Figure 46 A gene network diagram of tissue morphogenesis, striated muscle tissue development, muscle tissue development, and cardiac muscle tissue development divided by gene ontology terms is shown. The dot size for each term indicates the number of genes assigned to that term, with the smallest dot representing 20 genes and the largest dot representing 60 genes.

[0059] Figures 47A - 47B A quantitative set analysis of gene expression (QuSAGE) plot ( Figure 47A ) and the expression of genes related to REACTOME_SMOOTH_MUSCLE_CONTRACTION ( Figure 47B ) are shown. Compared to untransduced HDFs, the expression of genes related to smooth muscle contraction is significantly enriched in MATRA-treated HDFs (rSMCs).

[0060] Figures 48A - 48D A GuSAGE plot ( Figure 48A and 48C ) and REACTOME_SMOOTH_MUSCLE_CONTRACTION ( Figure 48B and 48D ) are shown, comparing the bulk RNA sequences in MYOCD-HDFs to untransduced HDFs ( Figure 48A -B) or MATRA-treated HDFs (rSMCs) ( Figure 48C -D).

[0061] Figures 49A - 49F A gene set enrichment analysis (GSEA) plot is shown, demonstrating enriched gene sets related to SMCs in MATRA-treated HDFs (rSMCs). By expression relative to HDFs, for muscle system processes ( Figure 49A ), muscle contraction (Figure 49B )), myocyte development( Figure 49C ), contractile fiber( Figure 49D ), actin cytoskeleton( Figure 49E ), actin binding( Figure 49F ) genes were sorted. The black vertical line at the bottom of each graph indicates the presence of gene signatures. Normalized Enrichment Score (NES); statistical significance is False Discovery Rate (FDR).

[0062] Figures 50A - 50H Shows cardiomyocyte (CM) genes in rSMCs compared to untransduced HDFs (e.g., genes related to: cardiac cell development( Figure 50A ), positive regulation of cardiac contraction( Figure 50B ), actin binding( Figure 50C ), alpha-actin binding( Figure 50D ), sarcomere organization( Figure 50E ), sarcoplasm( Figure 50F ), sarcoplasmic reticulum membrane( Figure 50G ) and I band( Figure 50H )) GSEA plots.

[0063] Figures 51A - 51F Shows a graph of qRT-PCR analysis of six genes specific to CM (TNNI1( Figure 51A ), TNNT2( Figure 51B ), TNNI3( Figure 51C ), MYH6( Figure 51D ), ACTN2( Figure 51E ) and MYH7( Figure 51F ). Expression of each gene was reduced in rSMCs, MYOCD-HDFs and untransduced HDFs compared to human embryonic stem cell-derived cardiomyocytes (hESC-derived CMs) on day 30. GADPH was used for normalization.

[0064] Figure 52A and Figure 52B Show laser Doppler perfusion images (LDPI)( Figure 52A ) and perfusion rate charts( Figure 52B)。Left leg, non-ischemic; right leg, ischemic; lighter gray, high LDPI index; darker gray, low LDPI index. In the graph, the perfusion rates depicted from bottom to top are HLI, HLI+HDF, HLI+MYOCD-HDF, and HLI+rSMC. The images and data show that rSMC treatment enhances blood flow in the ischemic limb.

[0065] Figure 53 Confocal microscopic images of longitudinal sections of ischemic limbs perfused with labeled isolectin B4 (ILB4) and harvested on day 28 after HLI are shown. rSMC enhanced blood flow into the hindlimb muscles in a murine hindlimb ischemia model compared to untreated or treatment with MYOCD-HDF or untransduced HDF. Fluorescence intensity represents the perfusion of fluorescein-conjugated ILB4 into the blood vessels of the hindlimb muscles.

[0066] Figure 54 Confocal images of ILB4-perfused ischemic hindlimbs harvested on day 28 after surgery (upper left), and its images after running AngioTool software after human rSMC transplantation (upper right) are shown. The bottom images are magnifications of the AngioTool images, showing the vessel surface, vascular structure, small non-vascular particles, and branch points.

[0067] Figure 55 The mean vascular density in the treatment groups of the murine ischemia model as described above for Figure 52 is shown. Treatment with rSMC significantly increased the mean vascular density compared to treatment with MYOCD-HDF or untransduced HDF. Vascular density is measured as the percentage of the vascular area divided by the explant area.

[0068] Figure 56 The mean vascular density in the treatment groups of the murine ischemia model after human rSMC transplantation is shown. In the murine hindlimb ischemia model, treatment with human rSMC significantly increased the vascular density compared to treatment with MYOCD-HDF or untransduced HDF. Vascular density is measured as Figure 55 described above.

[0069] Figure 57 The mean total vascular length of the treatment groups as described above for Figure 52 is shown. Treatment with rSMC significantly increased the mean total vascular length compared to treatment with MYOCD-HDF or untransduced HDF.

[0070] Figure 58 The mean total number of endpoints of the treatment groups as described above for Figure 52 is shown. Treatment with rSMC significantly increased the mean total number of vascular endpoints compared to treatment with MYOCD-HDF or untransduced HDF.

[0071] Figure 59Shows the total number of junctions for the treatment group described in Figure 52. Treatment with rSMCs significantly increased the average total number of vascular junctions compared to treatment with MYOCD-HDF or untransduced HDFs.

[0072] Figure 60 Shows the average junction density for the treatment group described in Figure 52. Treatment with rSMCs significantly increased the average vascular junction density compared to treatment with MYOCD-HDF or untransduced HDFs.

[0073] Figure 61 Shows the total vascular length of the treatment groups in the murine ischemia model after human rSMC transplantation. Treatment with rSMCs significantly increased the vascular density compared to treatment with MYOCD-HDF or untransduced HDFs.

[0074] Figure 62 Shows the total number of endpoints for the treatment groups in the murine ischemia model after human rSMC transplantation. Treatment with rSMCs significantly increased the total number of endpoints compared to treatment with MYOCD-HDF or untransduced HDFs.

[0075] Figure 63 Shows the total number of vascular junctions for the treatment groups in the murine ischemia model after human rSMC transplantation. Treatment with rSMCs significantly increased the total number of vascular junctions compared to treatment with MYOCD-HDF or untransduced HDFs.

[0076] Figure 64 Shows the junction density for the treatment groups in the murine ischemia model after human rSMC transplantation. Treatment with rSMCs significantly increased the vascular junction density compared to treatment with MYOCD-HDF or untransduced HDFs.

[0077] Figure 65 Shows the limb loss score for the treatment groups described in Figure 52. The limb loss score was calculated by the following scale to assess post-ischemic hindlimb injury: 0 = no necrosis; 1 = tip necrosis; 2 = toe necrosis; 3 = foot necrosis; 4 = leg necrosis; and 5 = whole limb loss. Treatment with rSMCs significantly reduced the limb loss score compared to treatment with MYOCD-HDF or untransduced HDFs.

[0078] Figure 66 Shows a hindlimb photograph of a representative animal with left hindlimb ischemia in the treatment groups of the murine ischemia model after human rSMC transplantation. Treatment with rSMCs significantly reduced loss or necrosis of the murine hindlimb compared to treatment with MYOCD-HDF or untransduced HDFs.

[0079] Figure 67It is shown that a strategy for differentiating lymphatic vessels from blood vessels determines functional endothelium by systemic injection of fluorescein-conjugated ILB4. Immunofluorescence staining of LYVE1 was performed on the ILB4-perfused ischemic hindlimbs harvested on the 28th day after surgery. The magnified images show that ILB4 is only restricted in expression in blood vessels and is not restricted in expression in - LYVE1 + lymphatic vessels. DAPI was used as a nuclear counterstain.

[0080] Figure 68 Figure is a schematic diagram of a sectioning strategy for visualizing a cross-section of an ischemic hindlimb after intramuscular transplantation of pre-labeled CM-DiI rSMCs and harvesting on the 28th day after surgery.

[0081] Figure 69 It is shown according to Figure 68 a confocal microscopic image of a cross-section of an ischemic hindlimb obtained. Arrows indicate ILB4 + surrounded by DiI + rSMC-labeled capillaries.

[0082] Figure 70 Figure is a schematic diagram of a sectioning strategy for visualizing a longitudinal section of an ischemic hindlimb after intramuscular transplantation of pre-labeled CM-DiI rSMCs and harvesting on the 28th day after surgery.

[0083] Figure 71 It is shown as according to Figure 70 a confocal microscopic image of a longitudinal section of an ischemic hindlimb obtained. rSMCs (arrows) indicate DiI + rSMCs associated with ILB4 + blood vessels.

[0084] Figure 72 It is shown the ACTA2 immunofluorescence staining of a cross-section of an ILB4 + arteriolar blood vessel. Arrows in the lower left panel indicate ILB4+ blood vessels surrounded by DiI+rSMCs or DiI + rSMC investment of ILB4 + arteriolar blood vessels. ACTA2 + rSMCs are located around the ILB4 + arteriolar blood vessels (curved dashed line in the upper right panel).

[0085] Figure 73 It is shown the SMTN immunofluorescence staining in a longitudinal section of a large ILB4 + blood vessel. Arrows in the lower left panel indicate ILB4 + blood vessels surrounded by DiI + rSMCs or DiI +rSMC coating of large ILB4 + Coating of blood vessels. DiI+rSMC express SMTN and are located in the blood vessel wall with a diameter of approximately 35 μm.

[0086] Figure 74 Shows SMTN labeled by immunofluorescence + DiI + Large ILB4 composed of rSMC + 3D rendering of blood vessels.

[0087] Figure 75 Shows DiI in the cross-section of the ILB4-perfused hindlimb harvested on the 28th day + Confocal microscopic images of HDF. DiI-prelabeled HDF was intramuscularly injected into three sites of the ischemic hindlimb.

[0088] Figure 76 Shows ILB4 + Immunofluorescence staining of ACTA2 in the longitudinal section of ILB4 blood vessels. The enlarged image shows DiI + Contribution of HDF to ILB4 + Capillaries, rather than to ILB4 + Contribution of large blood vessels. ACTA2+HDF is located around ILB4 + Capillaries, rather than around large blood vessels.

[0089] Figure 77 Shows ILB4 + Immunofluorescence staining of SMTN in the longitudinal section of ILB4 blood vessels. DiI-prelabeled rSMC was intramuscularly injected into three sites of the ischemic hindlimb. The enlarged image shows SMTN + DiI + rSMC (arrow) coating of ILB4 + Capillaries.

[0090] Figure 78 Shows ILB4 + Immunofluorescence staining of SMTN in the longitudinal section of ILB4 blood vessels. DiI-prelabeled rSMC was intramuscularly injected into three sites of the ischemic hindlimb. The enlarged image shows SMTN + DiI + rSMC (arrow) coating of ILB4 + Larger blood vessels.

[0091] Figure 79 Is a schematic diagram of the experimental design for assessing the effect of rSMC on vascular permeability. Human umbilical vein EC (HUVEC) and rSMC were seeded on both sides of a semi-porous membrane, and FITC-dextran was diffused onto the membrane.

[0092] Figure 80 A cross-sectional view of the diffusion of FITC-dextran through a HUVEC monolayer inoculated on the luminal side or a co-culture model of HUVEC and human cells (HDF and rSMC) inoculated on the outer luminal side of a 24-well Transwell insert is shown.

[0093] Figure 81 Immunofluorescence staining of PECAM1 and CNN1 in a HUVEC monolayer or a co-culture model of HUVEC and human cells (left) and quantitative analysis of FITC-dextran permeability in a HUVEC monolayer or a co-culture model of HUVEC and human cells (right) are shown.

[0094] Figures 82A - 82J qRT-PCR analysis of the expression of four genes specific for angiogenesis (Angpt1 ( Figure 82A ), Fgf2 ( Figure 82B ), VEGFA ( Figure 82C ), and Hif1a ( Figure 82D ), eight genes specific for arteriogenesis (Ccl2 ( Figure 82E ), Ccr2 ( Figure 82F ), Tgfb1 ( Figure 82G ), PDGFB ( Figure 82H ), Csf1 ( Figure 82I ), Mmp2 ( Figure 82J ), MMP3 ( Figure 82K ), Mmp9 (( Figure 82L ), two genes specific for the Notch signaling pathway (Hey1 ( Figure 82M ) and Dil4 ( Figure 82N )), and two genes specific for SMC implantation and vascular function (Igf1 ( Figure 82O ) and Hgf ( Figure 82P )) in non-ischemic or ischemic tissues (HLI, HLI + HDF, and HLI + rSMC) harvested on day 7 after transplantation is shown. Gapdh was used as a housekeeping gene and used for normalization. Detailed Description of the Invention

[0095] The present disclosure provides a method for generating reprogrammed smooth muscle cells (rSMCs). Also provided are rSMCs and compositions comprising a population of rSMCs. Also provided is a method of treating ischemia in a subject in need thereof using the compositions described herein. The rSMCs and their compositions overcome the limitations of currently available methods for treating ischemia. The rSMCs or their compositions promote neovascularization of capillaries as well as neovascularization of larger blood vessels such as arteries and arterioles, and also promote the recruitment of mural cells to capillaries and larger blood vessels. Additionally, the rSMCs or their compositions can be used to effectively promote perfusion of ischemic tissues and organs.

[0096] Methods for generating reprogrammed smooth muscle cells

[0097] The present disclosure provides a method for generating reprogrammed smooth muscle cells (rSMCs; used interchangeably herein with MATRA-treated fibroblasts). The method includes culturing fibroblasts with all-trans retinoic acid (ATRA) under conditions for generating rSMCs from fibroblasts, wherein the fibroblasts are genetically modified to overexpress myocardin.

[0098] As used throughout, rSMCs refer to cells generated from fibroblasts using the methods described herein. rSMCs (also referred to herein as MATRA-treated HDFs) have certain biomarkers and functions, such as naturally occurring smooth muscle cells. For example, rSMC cells co-express CNN1 and SMTN in a non-striated pattern and contract in the presence of intracellular calcium. However, unlike naturally occurring smooth muscle cells, rSMCs contain a heterologous nucleic acid sequence encoding myocardin.

[0099] The fibroblasts used to generate rSMCs can be mammalian fibroblasts, including, for example, human fibroblasts. Optionally, the fibroblasts are postnatal fibroblasts, including neonatal or adult fibroblasts. Optionally, the fibroblasts used to generate rSMCs are human dermal fibroblasts. The fibroblasts are optionally from the same subject to be treated or from a different subject.

[0100] As used throughout, ATRA refers to (2E,4E,6E,8E)-3,7-dimethyl-9-(2,6,6-trimethylcyclohex-1-en-1-yl)nona-2,4,6,8-tetraenoic acid.

[0101] Optionally, the conditions for generating one or more rSMCs from one or more genetically modified fibroblasts include culturing the fibroblasts in the presence of ATRA for a sufficient period of time (e.g., at least two days). Optionally, the contacting step lasts for 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 days.

[0102] Fibroblasts can be genetically modified either in vitro or in vivo. The genetic modification can be carried out before or concurrently with ATRA treatment. Fibroblasts are genetically modified to overexpress myocardin, for example, by introducing a heterologous nucleic acid encoding myocardin into the fibroblasts. As used herein, introduction in the context of introducing a heterologous nucleic acid into a cell refers to the translocation of the heterologous nucleic acid sequence from outside the cell to inside the cell. In some cases, introduction refers to the translocation of the heterologous nucleic acid from outside the cell to inside the nucleus.

[0103] A variety of translocation methods are contemplated, including but not limited to viral infection, transfection, transduction, electroporation, nanoparticle delivery, contact with nanowires or nanotubes, receptor-mediated endocytosis, translocation via cell-penetrating peptides, liposome-mediated translocation, or any method now known or hereafter identified for introducing nucleic acids into prokaryotic or eukaryotic cell hosts.

[0104] In some cases, the method of translocation is viral infection, such as using a viral vector. Examples of viral vectors include retroviral, lentiviral, adenoviral, and adeno-associated virus (AAV) vectors. In some cases, such as with adenoviral and AAV vectors, the vector does not integrate into the genome of the fibroblasts. In other cases, such as with retroviral and lentiviral vectors, the vector can integrate into the genome of the fibroblasts.

[0105] Targeted nuclease systems (e.g., RNA-guided nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), or megaTALs (MTs)) (see, e.g., Li et al., Signal Transduct. Target. Ther. 5(1):1 (2020)) can also be used to introduce a heterologous nucleic acid, such as a heterologous nucleic acid encoding myocardin, into fibroblasts.

[0106] The CRISPR / Cas9 system, an RNA-guided nuclease system employing the Cas9 endonuclease, can be used to modify genomic DNA in fibroblasts, for example, by inserting a heterologous nucleic acid sequence encoding myocardin into the fibroblasts. As used throughout, the CRISPR / Cas system refers to a class of bacterial systems widely used for defense against foreign nucleic acids. CRISPR / Cas systems are present in a wide range of eubacterial and archaeal organisms. CRISPR / Cas systems include type I, type II, and type III subtypes. The wild-type type II CRISPR / Cas system utilizes an RNA-mediated nuclease (e.g., Cas9), complexed with a guide RNA and an activator RNA, to recognize and cleave foreign nucleic acids. Guide RNAs having the activity of both a guide RNA and an activator RNA are also known in the art. In some cases, such dual-activity guide RNAs are referred to as single-guide RNAs (sgRNAs).

[0107] As used herein, Cas9 refers to an RNA-guided nuclease (e.g., a nuclease from a bacterium or archaea or a nuclease derived therefrom). Exemplary RNA-guided nucleases include the foregoing Cas9 proteins and their homologs. Other RNA-guided nucleases include Cpf1 (see, e.g., Zetsche et al., Cell 163(3):759-771 (2015)) and its homologs.

[0108] Cas9 homologs are present in a variety of eubacteria, including but not limited to bacteria of the following taxa: Actinobacteria, Aquificae, Bacteroidetes-Chlorobi, Chlamydiae-Verrucomicrobia, Chloroflexi, Cyanobacteria, Firmicutes, Proteobacteria, Spirochaetes, and Thermotogae. An exemplary Cas9 protein is the Streptococcus pyogenes Cas9 protein. Additional Cas9 proteins and their homologs are described, for example, in Chylinski et al., RNA Bio. 10(5):726-737 (2013); Hou et al., Proc. Natl. Acad. Sci. 110(39):15644-15649 (2011); and Sampson et al., Nature 497(7448):254-257 (2013). Variants of any of the Cas9 nucleases provided herein can be optimized to obtain efficient activity or enhanced stability in fibroblasts. Thus, engineered Cas9 nucleases are also contemplated. In some cases, engineered Cas9 is engineered such that the endonuclease domain (i.e., dCas9) is inactive. See, e.g., Chakraborty et al., Stem Cell Reports, 3(6):940-947 (2014); Black et al., Cell Stem Cell, 19(3):406-414 (2016); Rubio et al., Sci. Rep., 6:37540 (2016); Liu et al., Cell Stem Cell, 23:758-771 (2018); Wang et al., Acta. Pharm. Sin. B., 10(2):313-326 (2020); and Jiang et al., Mol. Ther., 30:54-74 (2022).

[0109] A heterologous nucleic acid encoding myocardin can be, for example, SEQ ID NO:71 or a nucleic acid sequence having at least 85%, 90%, 95% or 99% identity to a nucleic acid sequence comprising or consisting of SEQ ID NO:71.

[0110] Percent sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the polynucleotide sequence portion in the comparison window may include additions or deletions (i.e., gaps) as compared to the reference sequence (e.g., SEQ ID NO:71 or SEQ ID NO:72) for the optimal alignment of the two sequences which does not include additions or deletions. The percentage is calculated by determining the number of positions at which the identical nucleic acid bases occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percent sequence identity.

[0111] In the context of two or more nucleic acids, the term identical or percent identity refers to two or more sequences that are the same sequence. Two sequences are substantially identical if they have a specified percentage (e.g., 85%, 90%, 95% or 99%) of identical nucleotides when compared and aligned for maximum correspondence over a comparison window or specified region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

[0112] For sequence comparison, typically one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. Then the sequence comparison algorithm calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters.

[0113] As used herein, a comparison window includes any segment of a number of contiguous positions selected from the group consisting of: 50 to 600, typically about 75 to about 200, more typically about 100 to about 150, wherein after two sequences are optimally aligned, one sequence can be compared to the reference sequence for the same number of contiguous positions. Methods of sequence alignment for comparison are well known in the art.

[0114] The algorithms used to determine percent sequence identity and sequence similarity are the BLAST 2.0 algorithms, for example, as described in Altschul et al., J. Mol. Biol., 215:403-410 (1990). Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold. These initial neighborhood word hits act as seeds for initiating a search to find longer HSPs that contain them. The word hits are extended in both directions along each sequence until the cumulative alignment score can no longer increase. For nucleotide sequences, parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for a mismatched residue; always <0) are used to calculate the cumulative score. Extension of the word matches in each direction stops when: the cumulative alignment score drops by the quantity X from its maximum achieved value; the cumulative score becomes zero or less due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 11, an expectation value (E) of 10, M = 5, N = -4, and comparison of both strands as defaults.

[0115] As used throughout the text, the term nucleic acid or nucleotide refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) in single-stranded or double-stranded form, and polymers thereof. Unless explicitly restricted, the term encompasses nucleic acids containing known analogs of natural nucleotides that have similar binding properties as the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Those skilled in the art will recognize that a particular nucleic acid sequence can be modified to encompass its conservatively modified variants (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences, while retaining the function of the reference sequence (in this case, the sequence encoding myocardin). Any of the nucleic acid sequences described herein can be codon optimized.

[0116] As used herein, myocardin (SEQ ID NO:72) is a protein encoded by the MYOCD gene. As used throughout the text, the terms polypeptide, peptide, and protein are used interchangeably herein to refer to a polymer of amino acid residues. The term encompasses amino acid chains of any length, including full-length proteins, wherein the amino acid residues are linked by covalent peptide bonds.

[0117] As used herein, a gene is a DNA segment that participates in producing or encoding a polypeptide chain. It may include regions before and after the coding region (e.g., leader and trailing sequences) and intervening sequences (e.g., introns) between individual coding segments (exons).

[0118] As used throughout the text, heterologous refers to a substance that is not normally present in nature. For example, a heterologous nucleotide sequence is a nucleotide sequence that is not normally present in a given cell in nature. Thus, a heterologous nucleotide sequence can be foreign to its host cell (i.e., exogenous to the cell); naturally present in the host cell (i.e., endogenous), but present in the cell in an unnatural amount (i.e., more or less than the amount naturally present in the host cell); or naturally present in the host cell but located outside its natural locus.

[0119] Reprogrammed smooth muscle cells

[0120] The present disclosure provides an rSMC. Optionally, the rSMC is prepared by any one of the methods described herein; however, the rSMC can be a progeny of an rSMC that has been genetically modified to express or overexpress a heterologous myocardin-encoding nucleic acid and treated with ATRA. The rSMC contains a heterologous nucleic acid sequence encoding myocardin. Optionally, the rSMC cells co-express CNN1 and SMTN in a non-striated pattern. Smooth muscle cells lack sarcomeres and thus do not have striations like cardiomyocytes and skeletal muscle cells. In the presence of intracellular calcium, smooth muscle cells contract using the interaction of actin and myosin filaments.

[0121] The rSMC also contains and expresses one or more genes present in naturally occurring smooth muscle cells (e.g., CNN1, Calponin, and SMTN). As used herein, CNN1 is a gene encoding calponin 1, a protein thought to regulate actin filaments in smooth muscle cells. SMTN is a gene encoding Smoothelin, a protein marker of fully differentiated smooth muscle cells.

[0122] Optionally, in the presence of a sufficient amount of a vasoactive agent, the rSMC provided herein contracts by more than 10% (including, for example, more than 20% or more than 25%). The vasoactive agent can be selected from the group consisting of carbachol, endothelin-1, or potassium chloride. A sufficient amount of the vasoactive agent is an amount sufficient to release intracellular calcium in the rSMC and is an amount that promotes contraction of naturally occurring smooth muscle cells. Contraction can be measured, for example, by a change in cell surface area of more than 10% (including, for example, at least 11%, 12%, 15%, 20%, or 25%). Notably, the vasoactive agent reduces the surface area of untransduced HDFs by less than 10%. The contraction of the rSMC in response to the vasoactive agent is greater than the contraction visible in untransduced HDFs.

[0123] Compositions comprising reprogrammed smooth muscle cells

[0124] The rSMCs described herein can be formulated into a pharmaceutical composition. Optionally, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier. As used throughout the text, a carrier is a compound, composition, substance, or structure that, when combined with a compound or cell, aids or facilitates the preparation, storage, administration, delivery, effectiveness, selectivity, or any other characteristic of the cell to achieve its intended use or purpose. For example, a carrier can be selected to minimize any degradation of the cell and to minimize any adverse side effects after introducing the composition into a subject. Such pharmaceutically acceptable carriers include sterile biocompatible pharmaceutical carriers, including but not limited to saline, buffered saline, dextrose, and water. Pharmaceutically acceptable means that the material is not biological or otherwise undesirable and can be administered to an individual together with the selected agent without causing unacceptable biological effects or interacting in a harmful manner with other components of the pharmaceutical composition containing it.

[0125] The rSMCs can be formulated into a pharmaceutical composition for parenteral administration or local administration (e.g., intramuscular) at or near the ischemic site. In some instances, the pharmaceutical composition further comprises a second therapeutic agent, including an agent that directly or indirectly promotes perfusion, such as an angiotensin-converting enzyme (ACE) inhibitor, an angiotensin II receptor blocker (ARB), an antiplatelet agent, a nitrate, a β-blocker, a calcium channel blocker, or an anticoagulant.

[0126] Therapeutic methods

[0127] Also provided is a method of treating a subject suffering from ischemia or at risk of developing ischemia. The method comprises administering to the subject an effective amount of the rSMCs described herein; the population of reprogrammed smooth muscle cells described herein; or the pharmaceutical composition described herein.

[0128] As used herein, ischemia refers to a vascular disorder in which there is a reduction in blood supply to an organ, tissue, or part of the body. Ischemia can be caused by atherosclerotic blockage of blood vessels, such as peripheral artery disease, coronary artery disease, stroke, or heart attack. Ischemia can be characterized by low blood circulation and ultimately tissue necrosis. Ischemia is reduced by generating collateral blood supply and neovascularization, thereby allowing reperfusion of the organ, tissue, or body part. For example, ischemic diseases can affect the extremities, fingers, muscles, heart, liver, brain, etc.

[0129] As used throughout the text, a subject can be a vertebrate, more specifically a mammal (e.g., human, horse, cat, dog, cow, pig, sheep, goat, mouse, rabbit, rat, and guinea pig). The term does not denote a specific age or gender. Thus, it is intended to cover adult, neonatal, and pediatric subjects, whether male or female. As used herein, patient or subject can be used interchangeably and can refer to a subject diagnosed with a disorder or at risk of developing a disorder. The terms patient or subject include human and veterinary subjects. In any method provided herein, the subject can be a subject diagnosed with ischemia or at risk of developing ischemia.

[0130] The fibroblasts derived from rSMCs can be from the same subject to be treated (i.e., for autologous cell transplantation), or can be derived from a different donor (i.e., for allogeneic cell transplantation). Allogeneic cells can optionally be from a genetically related donor. Allogeneic transplantation may require immunosuppressive therapy, which can optionally be discontinued after neovascularization or reperfusion has occurred.

[0131] As used herein, the term treatment (treatment / treat / treating) refers to a method of reducing one or more effects of a disorder (e.g., ischemia in a subject) or one or more symptoms of a disorder. Thus, in the disclosed methods, treatment can refer to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the severity of ischemia. For example, if one or more symptoms of ischemia in a subject are reduced by 10% compared to a control, the method for treating ischemia is considered a treatment. Thus, the reduction can be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any percentage reduction between 10% and 100% compared to an untreated or control level. It should be understood that treatment does not necessarily mean a cure or complete elimination of the disorder or its symptoms.

[0132] As used herein, administer (administer / administration) refers to the act of introducing, injecting, or otherwise physically delivering a substance that exists in vitro (e.g., rSMCs, or cells differentiated therefrom) to a subject, such as by intracardiac, intravenous, intramuscular, rectal, oral, subcutaneous delivery, and / or any other physical delivery method described herein or known in the art. When treating a disease or its symptoms, the administration of the substance typically occurs after the onset of the disease or its symptoms. When preventing a disease or its symptoms, the administration of the substance typically occurs before the onset of the disease or its symptoms.

[0133] In the treatment methods described herein, a cell, cell population, or pharmaceutical composition is administered in an effective amount. As used herein, the term effective amount or therapeutically effective amount refers to the amount of a composition comprising any rSMC or a cell differentiated therefrom, which, when administered to a subject, is effective to treat a disease or disorder, either alone or in combination with additional agents, by one or more doses of a course of treatment. Suitable doses can depend on a variety of factors, including the particular rSMC or cell differentiated therefrom that is used and whether they are used in combination with other therapeutic agents. Other factors that can affect the dose administered to a subject include, for example, the type or severity of the disease.

[0134] Exemplary amounts of an effective amount of rSMC or a cell differentiated therefrom can be determined by one of ordinary skill in the art. Factors that can affect the dose can include, for example, other medical conditions that simultaneously or previously affected the subject, the overall health of the subject, the genetic predisposition of the subject, diet, the time of administration, and any other additional treatment regimens administered to the subject. It should also be understood that the specific dose and treatment regimen for any particular subject also depends on the judgment of the treating physician. A therapeutically effective amount is also an amount in which any toxic or detrimental effects of the composition are outweighed by the therapeutic beneficial effects.

[0135] Optionally, the effective amount increases the vascular perfusion, neovascularization, and / or arteriogenesis (i.e., the formation of new or larger blood vessels, such as arteries and arterioles) of the subject. For example, the effect can be measured by laser Doppler perfusion imaging.

[0136] Additional Definitions

[0137] As used in this specification and the appended claims, the singular forms "a / an" and "the" include plural referents unless the context clearly indicates otherwise.

[0138] The use of any and all examples or exemplary language (e.g., "such as") provided herein is for the purpose of better illustrating the invention only and does not impose a limitation on the scope of the invention unless otherwise claimed.

[0139] Unless the context clearly indicates otherwise, the terms "may", "may be", "can", and "can be" and related terms are intended to convey that the subject involved is optional (i.e., the subject occurs in some instances but not in others), rather than referring to the ability or possibility of the subject.

[0140] Unless otherwise indicated herein, the recitation of ranges of values herein is merely intended to be a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a concentration range is specified as 1% to 50%, values such as 2% to 40%, 10% to 30%, or 1% to 3% are intended to be expressly recited in the specification. These are merely examples of what is specifically intended, and all possible numerical combinations between and including the recited minimum value and the recited maximum value are to be considered to be expressly stated in this disclosure.

[0141] Examples

[0142] The following examples are set forth below to illustrate the compositions, methods, and results in accordance with the disclosed subject matter. These examples are not intended to include all aspects of the disclosed subject matter, but rather to illustrate representative methods, compositions, and results. These examples are not intended to exclude equivalents and variations of the disclosure, which will be apparent to those skilled in the art.

[0143] Example 1: Generation of Smooth Muscle-Like Cells by Direct Reprogramming Method

[0144] Materials and methods

[0145] Cell Culture and Maintenance. Human dermal fibroblasts (HDFs) were isolated from the dermis of human juvenile foreskin and amplified in Dulbecco's Modified Eagle Medium (DMEM) (Lonza, Basel, SWI) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, St. Louis, MO), 1X antibiotic-antimycotic (Anti-Anti) (Gibco, Waltham, MA), 1X MEM non-essential amino acids (MEM NEAA) (Gibco, Waltham, MA), and 1X GlutaMAX supplement (GlutaMAX) (Gibco, Waltham, MA) at 37 °C and 5% CO2. The Platinum-A (Plat-A) retroviral packaging cell line (Cell Biolabs, San Diego, CA) was maintained in the same medium without Anti-Anti and used for transfection from passage 4 to passage 7. HDFs and directly reprogrammed smooth muscle cells (rSMCs) were maintained in DMEM containing low glucose (HyClone, Logan, UT) supplemented with 5% FBS, Anti-Anti, MEM NEAA, and GlutaMAX.

[0146] Generation of retroviruses. The retroviral construct was generated by subcloning human MYOCD complementary deoxyribonucleic acid (cDNA) into the retroviral vector pMX. The construct was transfected into Plat-A cells using FuGENE HD (Promega, Madison, WI) according to the manufacturer's instructions. Viral supernatants were collected on days 2, 4, and 6 after transfection and filtered through a 0.45 μm polyethersulfone (PES) membrane filter (Corning, Corning, NY). The retroviruses were titrated using the Retro-X qRT-PCR Titration Kit (Takara Bio, Shiga, JP) according to the manufacturer's instructions.

[0147] Generation of directly reprogrammed SMCs. To directly reprogram HDFs into contractile SMCs, HDFs were seeded at a density of 1.5 x 10 5 cells / ml in DMEM (Lonza) supplemented with 10% FBS (Sigma-Aldrich), Anti-Anti (Gibco), MEM NEAA (Gibco), and GlutaMAX (Gibco) at 37 °C and 5% CO2. Cells were infected overnight with filtered retroviral medium containing 4 μg / ml polybrene (Sigma-Aldrich) with or without 0.4, 2, or 10 μM all-trans retinoic acid (ATRA) (Sigma-Aldrich) per liter, and the viral medium was replaced with DMEM / low glucose (HyClone) supplemented with 5% FBS, Anti-Anti, MEM NEAA, and GlutaMAX with or without ATRA for 24 h. The viral infection was repeated twice, and the cells were maintained in DMEM / low glucose containing 5% FBS with or without ATRA for the duration of the culture.

[0148] Quantitative reverse transcription polymerase chain reaction (qRT-PCR). Total ribonucleic acid (RNA) was isolated from cells using the RNeasy Mini Kit (QIAGEN, Hilden, GE) or TRIsure (Bioline, Memphis, TN) according to the manufacturer's instructions. The extracted RNA was reverse transcribed using TaqMan Reverse Transcription Reagent (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. The synthesized cDNA was amplified using PowerUP SYBR Green Master Mix (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions, and qRT-PCR was performed using human or mouse specific primers as shown in Table 1. The RNA levels were quantitatively evaluated using a QuantStudio 3 96-well 0.2-ml real-time PCR system (Applied Biosystems). The relative messenger RNA (mRNA) expression was normalized to GAPDH.

[0149] Table 1. Primers used for qRT-PCR analysis in Example 1

[0150]

[0151]

[0152] Flow cytometry. Cells were washed with Dulbecco's phosphate-buffered saline (DPBS; Corning), detached with ACCUTASE (STEMCELL Technologies, Vancouver, BC), and harvested with autoMACS running buffer (Miltenyi Biotec, Bergisch Gladbach, GE). Cells were fixed and permeabilized using the Cytofix / Cytoperm Fixation / Permeabilization Kit (BD Biosciences, Franklin Lakes, NJ) according to the manufacturer's instructions. The cells were then incubated overnight at 4 °C in the dark with unconjugated primary antibodies followed by incubation with fluorescent dye-conjugated secondary antibodies for at least three hours. The primary and secondary antibodies are shown in Table 2. Fluorescence-activated cells were analyzed by an LSR Fortessa flow cytometer (BD Biosciences). Flow cytometry data were analyzed using FlowJo TM v10.8 software (BD Biosciences).

[0153] Immunocytochemistry. Cells were washed with DPBS (Corning), fixed in 4% paraformaldehyde (VWR, Radnor, PA) for half an hour at room temperature in the dark, and permeabilized with 0.5 - 1% Triton X - 100 (Sigma - Aldrich) in DPBS for one hour at room temperature in the dark. The permeabilized cells were then incubated with blocking buffer containing 0.5 - 1% Triton X - 100 and 1% bovine serum albumin (Miltenyi Biotec) in DPBS for three hours at room temperature in the dark. According to the manufacturer's instructions, the cells were incubated overnight at 4°C in the dark with unconjugated primary antibodies, followed by incubation with fluorescent dye - labeled secondary antibodies for three hours at 4°C in the dark. The primary and secondary antibodies are shown in Table 2. Cells were counterstained with DAPI (Invitrogen, Carlsbad, CA) to visualize the nuclei. Images were captured by a Zeiss LSM 700 or 780 confocal microscope (Carl Zeiss AG, Oberkochen, GE).

[0154] Table 2. Antibodies

[0155]

[0156]

[0157] Results

[0158] ATRA alone only slightly induced SMC genes in HDFs. As Figure 1 shown, HDFs were treated with different concentrations of ATRA for four days. The mRNA expression of STRA6, S100A4, and SMC genes (MYOCD, ACTA2, TAGLN, CALD1, DES, CNN1, MYH11, and SMTN) in ATRA - treated and untreated HDFs was examined by qRT - PCR. As Figure 2 shown in - 3, compared with untreated HDFs, the RNA expression of STRA6 and SMC genes increased in ATRA - treated HDFs. The expression of S100A4 decreased significantly( Figure 4 ). Among the ATRA concentrations tested, 2 μM was the most effective in inducing SMC gene expression (Figure 3). Overall, the fold increase in SMC gene expression ranged from 1.1 (CALD1) to 6.1 (MYH11) (Figure 3). These results indicate that ATRA alone only slightly induced SMC genes in HDFs.

[0159] Overexpression of the combination of MYOCD and ATRA induced robust SMC gene expression in HDFs. As Figure 5As shown, HDFs were infected twice with either the individual retrovirus MYOCD or in combination with 2 μM ATRA and cultured for 16 days. qRT-PCR showed that MYOCD-transduced HDFs expressed eight SMC genes as early as day 2, but mostly peaked at day 8 (Figure 6). The peak expression of MYOCD increased to 295.4-fold, while the expression of other SMC genes increased 2.2-fold (TAGLN, CALD1) to 29.2-fold (MYH11) (Figure 6). Notably, HDFs treated with ATRA and MYOCD (MATRA) (also referred to herein as reprogrammed SMCS or rSMC) showed a more robust expression of eight SMC genes, with other genes peaking at day 4 except for SMTN which peaked at day 8 (Figure 6). As shown in Figure 6, the fold increase in SMC gene expression ranged from 3.9 (CALD1) to 2639.2 (MYOCD). Although gradually decreasing over time, these contractile SMC genes were not silenced and mostly remained high within 16 days (Figure 6).

[0160] MYOCD and ATRA can increase the expression of contractile SMC and pericyte genes in HDFs. The expression of SMC genes in MATRA-treated HDFs mostly peaked at day 4 (Figure 6). In particular, MYOCD gradually decreased over time but remained higher in MATRA-treated HDFs than in untransduced HDFs and MYOCD-transduced HDFs (Figure 6). As Figure 7As shown, day 4 was chosen as the target date to characterize the reprogrammed SMCs. qRT-PCR showed that in MATRA-treated HDFs, the expression of MYOCD was significantly increased, 2,642.9-fold higher than that in untransduced HDFs and 10.0-fold higher than that in MYOCD-transduced HDFs (Figure 8). Compared with untransduced HDFs, other SMC genes and STRA6 were also significantly increased in MATRA-treated HDFs: 34.7-fold (ACTA2), 4.0-fold (TAGLN), 3.0-fold (CALD1), 4.6-fold (DES), 7.6-fold (CNN1), 51.4-fold (MYH11), 2.3-fold (SMTN), and 5.0-fold (STRA6) (Figures 8-9). Interestingly, the expression of three SMC genes (VCL, TPM1, and TPM2), which play important roles in regulating SMC contraction, was significantly increased only in MATRA-treated HDFs (Figure 10). On the other hand, the expression of four representative genes (KLF4, MYH10, MMP2, and SPP1), which are specific for synthetic SMCs, was decreased in MATRA-treated HDFs (Figure 11). Additionally, the expression of the representative pericyte gene CSPG4 was increased in both MYOCD-transduced and MATRA-treated HDFs compared with untransduced HDFs because of the existence of a continuous phenotypic transition from pericytes to vascular SMCs and vice versa ( Figure 12 ). Finally, the mRNA expression of four representative fibroblast genes, such as S100A4, THY1, VIM, and PDGFRA, was significantly decreased under this reprogramming condition (Figure 13). In summary, the results indicate that overexpression of MYOCD together with ATRA treatment can significantly increase the contractile SMC and pericyte genes in HDFs.

[0161] Protein analysis confirmed that the combination of ATRA and MYOCD can drive a stronger transition to the SMC phenotype in HDFs. The expression of SMC genes was confirmed at the protein level. Flow cytometry analysis showed that on day 4, MATRA-treated HDFs exhibited ACTA2 and MYH11 in approximately 57% and 48% of the cells, respectively ( Figures 14 - 15 ). The gating strategy for flow cytometry is shown in Figures 16 - 18 . Figure 16 The gating for selecting single live cells is shown. Figures 17 - 18 The gating for identifying ACTA2+ and MYH11+ cells is shown, respectively. As Figures 19 - 20As shown, immunocytochemistry further confirmed the expression of four SMC markers, ACTA2, TAGLN, CNN1, and SMTN. In untransduced HDFs, ACTA2, CNN1, and SMTN were barely detectable, with diffuse staining for TAGLN. In MYOCD-transduced HDFs, ACTA2 and TAGLN were clearly expressed, while CNN1 and SMTN were expressed limitedly in actin cytoskeleton organization. MATRA-treated HDFs robustly expressed ACTA2 and TAGLN and had a more prominent actin stress pattern. MATRA-treated HDFs co-expressed CNN1 and SMTN in a typical non-striated SMC-like pattern. Although overexpression of MYOCD was essential for direct reprogramming of HDFs into SMCs, addition of ATRA showed a synergistic effect on induction of contractile SMC genes. Collectively, these data indicate that the combination of MYOCD and ATRA can drive a more robust change in cell fate towards a contractile SMC phenotype.

[0162] Example 2: Contractile characteristics of rSMCs

[0163] Materials and methods

[0164] Contractility assessment. HDFs were cultured and directly reprogrammed into SMCs in the same manner as above. Cells were treated with 100 μM carbachol (Sigma-Aldrich) for approximately 10 minutes. Cells were seeded onto glass-bottom dishes (Thermo Fisher Scientific). Contraction was monitored at the cell level, and time series were acquired at a rate of one frame every 30 seconds for 15 minutes using a Zeiss LSM 700 or 780 confocal microscope (Carl Zeiss AG) equipped with a Zeiss stage microscope incubation system. Changes in cell surface area before and after addition of carbachol were evaluated using the modular image acquisition, processing, and analysis software ZEISS Efficient Navigation (blue edition) (ZEN (blue edition); Carl Zeiss AG).

[0165] Collagen gel contraction assay. Cells were washed with DPBS (Corning), detached with 0.25% trypsin-EDTA (Gibco), and resuspended at 1.5x 10 5The cells were resuspended at a density of 0.4 ml in the medium. The collagen lattice was prepared by mixing the cell suspension with 0.2 ml of rat tail type I collagen (3 mg / ml; Gibco) and rapidly transferring it to a 4-well plate after adding an appropriate amount of 1 molar / liter sodium chloride (Sigma-Aldrich). The collagen gel was polymerized at room temperature for 20 minutes, separated from the wells, and incubated at 37 °C and 5% CO2 for two days. The change in the gel diameter was captured by a digital camera and evaluated using ImageJ (National Institutes of Health).

[0166] Detection of calcium release. The cells were washed with DPBS (Corning) and pre-loaded with the calcium-sensitive fluorescent dye Fluo-4, AM (Thermo Fisher Scientific) in Opti-MEM low-serum medium (Gibco) at 37 °C and 5% CO2 for one hour. The pre-loaded cells were then washed at 37 °C and 5% CO2 for 15 minutes to remove the intracellular acetoxymethyl ester. Using a Zeiss LSM 700 or 780 confocal microscope (Carl Zeiss AG), the changes in intracellular calcium release were acquired in a time series at a rate of one frame every 0.2 milliseconds for 550 seconds before and after adding 100 μM carbachol (Sigma-Aldrich). The pre-loaded cells were individually selected from the field of view, and the fluorescence intensity of Fluo-4 in individual cells was measured and normalized to the baseline (F / F0). The relative fluorescence units of the Fluo-4, AM intensity were analyzed using ZEN (blue edition) (Carl Zeiss AG).

[0167] TEM. Cells were washed with DPBS (Corning), detached with 0.25% trypsin-EDTA (Gibco), and pre-fixed overnight at 4 °C in the dark with Karnovsky fixative containing 2% glutaraldehyde (Merck, Darmstadt, GE), 2% PFA (Merck), and 0.5% calcium chloride (Sigma-Aldrich) in 0.1 mol / L PBS (Sigma-Aldrich). Cells were washed with PBS for two hours and then fixed with 1% osmium tetroxide (Polysciences, Warrington, PA) in PBS for two hours. Fixed cells were washed with PBS for 10 minutes and gradually dehydrated through a series of ethanol dilutions (Merck) increasing to absolute ethanol. Dehydrated cells were infiltrated with propylene oxide (Sigma-Aldrich) for 10 minutes and embedded with a Poly / Bed 812 (Luft formulation) embedding kit / DMP-30 (Polysciences). Embedded cells were then polymerized at 65 °C for 12 hours in a TD-700 electron microscope oven (DOSAKA, Kyoto, JP). Blocks were sectioned into 200-nm semi-thin sections using a diamond knife in an ultramicrotome Leica EM UC7 (Leica Microsystems, Wetzlar, GE), and sections were stained with toluidine blue (Sigma-Aldrich). Regions of interest were selected and sectioned into 80-nm thin sections using an ultramicrotome. Thin sections were then immersed in 3% uranyl acetate (Polysciences) for 30 minutes and in 3% lead citrate (Polysciences) for seven minutes and then captured at an accelerating voltage of 80 kV using a JEM-1011 transmission electron microscope (JEOL Ltd., Tokyo, JP) equipped with a Megaview III CCD camera (Soft Imaging System GmbH, Münster, GE).

[0168] Scratch wound healing assay. Cells were seeded onto 6-well plates at a density of 2.0 x 10 5 cells / mL and incubated overnight at 37 °C and 5% CO2. The confluent cell monolayer was scratched in a straight line using a p200 pipette tip to create a "scratch". Scratch fields were acquired before and 24 hours after using a phase contrast microscope. The area of scratch closure was measured using the Java-based image processing program ImageJ (National Institutes of Health) for further analysis of the images.

[0169] Results

[0170] Increased contractility in MYOCD- and MATRA-transduced HDFs. The most prominent feature of contractile SMCs is the ability to contract. Therefore, the contractility of MATRA-treated HDFs was determined after stimulation with the vasoconstrictor carbachol. After carbachol treatment, untransduced HDFs showed minimal changes in cell surface area, contracting by approximately 9%, while MYOCD-transduced HDFs contracted by approximately 20%( Figure 21 ). However, the contracting MATRA-treated HDFs exhibited a cell surface area change of approximately 28%, similar to that seen in human aortic SMCs (HAoSMCs) (approximately 32%)( Figure 21 ). To further confirm their contractility, a carbachol-induced collagen gel contraction assay was performed( Figure 22 ). Similar results were obtained in collagen gels embedded with these cells. MYOCD-transduced and MATRA-treated HDFs in the gels showed stronger contraction than HAoSMCs or untransduced HDFs in the gels( Figure 22 ).

[0171] Increased intracellular calcium release in MATRA-transduced HDFs. Changes in intracellular calcium (Ca 2+ ) are crucial for the contractile function of SMCs (see, e.g., Somlyo & Somylo, Nature, 460:705 - 710 (2009)). After carbachol treatment, untransduced HDFs exhibited low intracellular Ca 2+ release( Figure 23 ). MYOCD-transduced HDFs and MATRA-treated HDFs showed higher intracellular Ca 2+ release, which was similar to that of HAoSMCs( Figure 23 ). Quantitative analysis of Fluo-4 intensity showed that the response rate of MATRA-treated HDFs was significantly higher compared to other controls (MATRA, approximately 63%; HDF, approximately 11%; MYOCD-HDF, approximately 44%; HAoSMC, approximately 54%)( Figure 24 ). As Figure 25 shown, the maximum fluorescence intensity ((F / F0)max) and the time to reach the fluorescence peak (time to reach (F / F0)max) were determined. The (F / F0)max in response to carbachol in MATRA-treated HDFs was significantly higher compared to all other control groups( Figure 26 ). The time to reach (F / F0)max in MYOCD-transduced HDFs and MATRA-treated HDFs was significantly shorter compared to untransduced HDFs( Figure 27 ). Intracellular Ca 2+The temporal characteristics of the release are similar to those of HAoSMC. Monitoring F / F0 in individual cells over time further shows that individual cells in each group exhibit significant changes in the number of Ca 2+ events ([[]] Figures 28 - 31 ). The vast majority of untransduced HDFs do not produce or produce a large main peak after carbachol treatment, while MYOCD-transduced HDFs and HAoSMC usually have large main peaks followed by a small number of repetitive peaks ([[]] Figure 32 ). In contrast, MATRA-treated HDFs produced more robust main peaks and submaximal Ca 2+ events in the form of propagating waves over a longer period ([[]] Figure 32 ). Vasoconstrictor-induced propagating Ca 2+ events have been demonstrated in in situ or freshly isolated SMCs (see, e.g., Halaidych et al., Stem Cell Reports, 12:647-656 (2019); Blatter & Wier, Am. J. Physiol. 263(576-586) (1992); Gordienko et al., J. Physiol., 507(3)707-720 (1998); Borysova et al., Cell Calcium, 54(163-174) (2013)). Such events are reflected in MATRA-treated HDFs, indicating that their contractile properties are stronger than those of MYOCD-transduced HDFs.

[0172] In MATRA-treated HDFs, cell migration was reduced. Acquisition of the contractile SMC phenotype is usually accompanied by a reduction in cell migration, which was evaluated in this study by scratch wound healing assay. At 24 hours after scratching, the migration distance measured as the percentage of wound closure was significantly lower in MATRA-treated HDFs compared to untransduced and MYOCD-transduced HDFs ([[]] Figure 33 ).

[0173] The cellular ultrastructure of MATRA-treated HDFs showed characteristics of contractile SMCs. The cellular ultrastructure of the cytoskeleton and contractile apparatus of the cells was evaluated by transmission electron microscopy (TEM) ([[]] Figure 34 ). TEM showed that untransduced HDFs contained abundant mitochondria and free ribosomes, which are important characteristics of fibroblasts (see, e.g., Lucky et al., Exp. Cell Res. 92(383-393) (1975)), but did not have discrete contractile filaments ([[]] Figure 34 ). In contrast, MYOCD-transduced HDFs showed a reduction in mitochondria and free ribosomes and an increase in contractile filaments in the absence of striations ([[]] Figure 34 ). Notably, as Figure 34As shown, the MATRA-treated HDFs showed further increased contractile filaments, which were anchored by dense bodies (a key ultrastructural feature of contractile SMCs) in a typical non-striated SMC-like pattern (see, e.g., Ross, J. Cell Bio. 50:172-186 (1971)). In addition, there was a significant loss of mitochondria and ribosomes ( Figure 34 ).

[0174] Example 3: Transcriptome analysis demonstrating the reprogramming of HDFs into contractile SMCs

[0175] Materials and methods

[0176] Bulk RNA sequencing (RNA-seq) analysis. Total RNA was obtained from two biological replicates of each group (HAoSMC, HDF, MYOCD-only cells, and MATRA-treated cells) using the miRNeasy Mini kit (QIAGEN) according to the manufacturer's instructions. The integrity and concentration of the samples were evaluated by an Agilent 2100 BioAnalyzer (Agilent Technologies, Inc., Santa Clara, CA), and only samples with an RNA integrity number higher than 8 were used. Polyadenylated (poly(A)) mRNA was enriched by magnetic beads containing oligo(dT), and then fragmented into short pieces. According to the manufacturer's instructions, the cDNA was end-repaired and poly(A)-tailed using the TruSeq Stranded mRNA Sample Preparation kit (Illumina, Inc., San Diego, CA) and ligated to sequencing adapters. Then, the libraries with a size range between 120-200 base pairs (bp) were subjected to paired-end sequencing on an Illumina NovaSeq 6000 (Illumina, Inc.) platform with a read length of 150 bp, generating an average of 27 million reads per library, as shown in Table 3. The raw reads were quality-assessed, and only the clean reads of each sample were further analyzed.

[0177] Table 3. Sequencing data for bulk RNA-seq analysis

[0178]

[0179]

[0180] Bioinformatics analysis. Cutadapt (an open-source software developed by Marcel Martin and provided under the MIT license) was used to preprocess and filter the reads by removing low-quality reads and adapter sequences. The filtered reads were aligned to the reference genome of the Genome Reference Consortium Human Build 38 (GRCh38; hg38) using STAR (an open-source software developed by Alexander Dobin et al. under the GPLv3 license). Then, featureCounts (an open-source software developed by Yang Liao et al. and provided under the GNU General Public License) and a set of default parameters were used to estimate gene expression levels. DESeq2 (an open-source software developed by Michael Love et al. and available on the Bioconductor platform) was used for differential expression gene (DEG) analysis between groups, with the gene information cutoff set to p-value < 0.05 and absolute log fold change value > 2.0, as shown in Table 4. Additionally, iDEP.91 (a software developed by Steven Ge et al. and available on the Bioconductor platform) was used for bioinformatics analysis. Gene sets defined for HDF and rSMC for gene set enrichment analysis (GSEA) were defined by more than two-fold gene expression changes. The enrichment of these gene sets was evaluated using the GSEA software (v4.1.0; The Broad Institute, Cambridge, MA), with 1,000 permutations of the gene sets, no dataset collapse, and weighted enrichment statistics used.

[0181] Table 4. Differentially expressed genes in rSMC

[0182]

[0183]

[0184] Results

[0185] SMC gene expression in MATRA-treated HDF. To decipher the transition of cell fate during reprogramming, RNA-seq was performed using total RNA from HDF, MYOCD-only cells, rSMC, and HAoSMC. Principal component analysis (PCA) showed four distinct groups ( Figures 35 - 36 ). Although MYOCD-only cells showed a gene expression pattern similar to that of HDF, rSMC showed a distinct transcriptome pattern ( Figures 37 - 39 ). Hierarchical clustering analysis revealed that rSMC and HAoSMC were close in position and distinct from HDF and MYOCD-only cells ( Figure 38)。To further investigate the distinguishable gene expression patterns of these cell populations, soft clustering was applied to the RNA-seq data analysis. The results showed that more than 1000 genes were enriched in each population and approximately 600 to 2000 genes were downregulated (Figs. 40 - 41). As shown in Fig. 40, HDF highly expressed KIF4A, CDCA8, CENPA, and GTSE1, which are related to mitosis and cell proliferation (see, e.g., Ultera et al., EMBO J., 17:5015 - 5025 (1998); Howman et al., Proc. Natl. Acad. Sci., 97:1148 - 1153 (2000); Mazumdar & Misteli, J. Cell Bio., 166(613 - 620) (2004); Sampath et al., Cell, 118:187 - 202 (2004)). Only MYOCD cells expressed high levels of endolysosomal genes (LYPLA2, TPP1, and TANGO2) and cytoskeletal genes (EPS8L2 and SYNE3) (Fig. 40). rSMC were enriched for genes involved in muscle development and function (SORBS1, CNN1, and MYL7) and embryonic skeletal system morphogenesis (HOXD13 and HOXC13) (Fig. 40). Cultured HAoSMC highly expressed genes related to the signal transduction system (MRAP2, SULT1E1, CACNG8, and CD200) (Fig. 40). Gene ontology (GO) analysis was consistent with these results (Fig. 40). The expression of contractile SMC marker genes (ACTA2, CALD1, CNN1, MYH11, MYL6, MYOCD, TAGLN, TPM1, TPM2, and VCL) was further compared among the four groups, and rSMC showed significantly higher levels of these genes (Fig. 42). In summary, these RNA-seq analyses demonstrated that rSMC are enriched for SMC genes, particularly contractile genes.

[0186] rSMC display molecular features of the contractile SMC phenotype. To reconstruct the reprogramming from HDF to rSMC, differentially expressed genes (DEGs) between these two groups were identified by using a threshold of false discovery rate less than 0.1 and fold change greater than 2.0, as shown in Table 4. The genes most upregulated in rSMC were those encoding contractile and structural proteins of SMC (ACTA1 and 2, ACTG2, CNN1, MYH11, MYLK, and MYL7) and their SMC fate-determining transcription factor MYOCD ( Figure 43 ). GO terms enriched in rSMC were classified into angiogenesis, chemotaxis, embryonic skeletal system morphogenesis and development, striated and cardiac muscle tissue development, and muscle tissue development and contraction ( Figure 44 ). Genes involved in these terms are as Figures 45 - 46as depicted. Some of the enriched genes in rSMCs are ACTA1 and 2, ACTG2, MYH11, TPM1, MYLK, and MYL7 and 9, representing contractile SMCs( Figure 43 ). In addition, quantitative set analysis of gene expression (QuSAGE) was performed to reveal differentially expressed signaling pathways. Among these signaling pathways, smooth muscle contraction was specifically selected to measure the log2 fold change (logFC) between HDFs and rSMCs (Figure 47). The logFC was 245.7, significantly higher than the logFC between HDFs and MYOCD-only cells (13.8) and the logFC between MYOCD-only cells and rSMCs (231.9) (Figure 48). GSEA showed upregulation of genes in the muscle system process, muscle contraction, myocyte development, contractile fiber, actin cytoskeleton, and actin binding, all of which are related to the functions of contractile SMCs (Figure 49). GSEA further showed enrichment of cardiac genes (NKX2.5, TNNT1 and 2, GATA6, HAND1, and MYL7) and cardiac-related biological processes (cardiac cell development, positive regulation of cardiac contraction, actin binding, alpha-actin binding, sarcomere organization, sarcoplasm, sarcoplasmic reticulum, and I band) because MYOCD is a well-known cardiac transcription factor (see, e.g., Wang et al., Cell, 105:851-862 (2001)) (Figure 50). As shown in Figure 51, the expression of cardiomyocyte (CM) marker genes (TNNI1 and 3, ACTN2, TNNT2, and MYH6 and 7) was further examined by qRT-PCR and compared to hESC-derived CMs. Although MYOCD alone or in combination with ATRA slightly induced CM genes, their levels were quite low compared to hESC-CMs (Figure 51), and no other CM characteristics were shown. Overall, these findings further support that rSMCs display molecular characteristics of the contractile SMC phenotype.

[0187] Example 4: Effect of rSMCs on Tissue Ischemia Recovery

[0188] Materials and methods

[0189] Induction of hindlimb ischemia and cell transplantation. Hindlimb ischemia was induced in 8- to 10-week-old athymic male nude mice (Japan Shizouka Laboratory Center (SLC), Inc., Shizuoka, JP). The femoral artery was ligated and the major branches were cauterized. Then the mice were randomly assigned to four groups: surgery only (HLI), HDF injection group, MYOCD cell injection only group, or rSMC injection group (HLI+HDF, HLI+MYOCD, and HLI+rSMC). To determine the treatment effect, 2 x 10 5 cells in 100 μl of DPBS (Corning) were intramuscularly injected into three sites of the ischemic hindlimb. Before injection, the cells were pre-labeled with chloromethylbenzoyl amino (CellTrackerTM CM-DiI; DiI; Invitrogen, Carlsbad, CA) to monitor cell behavior in the tissue.

[0190] Measurement of blood flow in the hindlimb. Blood flow in the hindlimb was measured with a laser Doppler perfusion imager (Moor Instruments Ltd., Axminister, UK) after surgery and measured once a week for four weeks. The perfusion average was calculated from the stored digital color-coded images. The blood flow level of the ischemic limb was normalized to the non-ischemic limb to evaluate tissue function and avoid data variations caused by ambient light and temperature, as shown in Table 5.

[0191] Table 5. Blood perfusion measured by laser Doppler perfusion imaging

[0192]

[0193] Quantitative analysis of vascular function in ischemic tissues. The vascular function in ischemic tissues was quantitatively analyzed using AngioTool (open-source software developed by Zudaire et al., provided under the GNU General Public License). Four weeks after transplantation, mice (Japan SLC, Inc.) were first anesthetized and fluorescein-conjugated Griffonia simplicifolia lectin, isolectin B4 (ILB4; Vector Laboratories Inc., Burlingame, CA) was injected intravenously. The hindlimb muscles were removed, fixed overnight at 4°C in 4% PFA in the dark, and incubated overnight at 4°C in the dark in a 30% sucrose (Sigma-Aldrich) solution. The tissues were embedded in optimal cutting temperature (OCT) compound (Sakura Finetek USA, Torrance, CA) and sectioned into slices with a thickness in the range of 8 to 50 μm using a Leica CM1860 cryostat (Leica Biosystems Nussloch GmbH, Nussloch, GE). Five to eight tissue sections with a thickness range of 25 to 30 μm were randomly selected from each animal, counterstained with DAPI (Invitrogen), and analyzed using a Zeiss LSM 700 or 780 confocal microscope (Carl Zeiss AG). Vascular function was then calculated from at least twenty randomly selected fields of view using AngioTool software.

[0194] Limb loss score index. Four weeks after inducing hindlimb ischemia, mice (Japan Shizuoka Laboratory Center (SLC), Inc., Shizuoka, Japan) were euthanized, and the ischemic limbs were photographed with a digital camera and evaluated by the following scoring to assess the ischemic hindlimb injury: 0 = no necrosis; 1 = tip necrosis; 2 = toe necrosis; 3 = foot necrosis; 4 = leg necrosis; and 5 = whole limb loss.

[0195] Results

[0196] rSMCs can enhance the recovery of hindlimb ischemia and promote neovascularization. To test whether rSMCs can promote the recovery of tissue ischemia, a murine hindlimb ischemia model was used. After ligation of the femoral vessels, HDFs, MYOCD-only cells, or rSMCs were directly injected into the ischemic thigh muscles. As shown in Figure 52, serial analysis of blood perfusion by laser Doppler perfusion imaging (LDPI) demonstrated that the flow recovery in the rSMC group was significantly enhanced compared with the HDF and MYOCD-only groups. This was evident 14 days after ischemia induction and persisted until day 28 (Figure 52). We sacrificed the mice on day 28 and harvested the muscles after injecting fluorescein-conjugated Griffonia (Bandeiraea) simplicifolia isolectin B4 (ILB4) into the heart for whole-body vascular perfusion. The density of ILB4+ vessels in the hindlimb muscles was significantly higher in the rSMC group compared with the other groups (HLI, HDF, and MYOCD-only) ( Figure 53 ). To analyze the vascular network, confocal images of ILB4+ vessels were evaluated using AngioTool. These ILB4+ vessels and their branch points were marked ( Figure 54 ). Then, morphological parameters, including vessel density, vessel length, the number of end points and junctions, and junction density, were calculated. As Figures 55 - 64 shown, all of these parameters were significantly higher in the rSMC group compared with the other groups. These results indicate that injection of rSMCs not only increased the number of blood vessels but also increased their network formation. Additionally, the rSMC group showed a significantly lower limb loss score ( Figures 65 - 66 ). Collectively, these results indicate that rSMCs enhance the recovery of hindlimb ischemia and promote functional and structural neovascularization.

[0197] Example 5: Contribution of rSMCs to Microvascular Formation

[0198] Materials and methods

[0199] Histological analysis. Hindlimb ischemia and cell transplantation were performed as described above. The cells were labeled with CellTracker TMPre-labeled with CM-DiI and then injected into the ischemic hindlimb tissue. Twenty-eight days after injection, before euthanasia, the mice (Japan SLC, Inc.) were systemically perfused with fluorescein-conjugated ILB4 (Vector Laboratories, Inc.) to identify functional endothelium. The ischemic hindlimb tissue of the mice was removed, fixed overnight at 4 °C in 4% PFA (VWR), and incubated overnight at 4 °C in the dark in a 30% sucrose (Sigma-Aldrich) solution. Then, tissue sections of the ischemic hindlimb tissue were prepared. The tissue sections were washed with DPBS (Corning), fixed with 4% PFA for half an hour at room temperature in the dark, and permeabilized with 1-3% Triton X-100 (Sigma-Aldrich) in DPBS for one hour at room temperature in the dark. The permeabilized tissue sections were incubated with a blocking buffer containing 0.5-1% Triton X-100 and 1% BSA in DPBS for three hours at room temperature in the dark. According to the manufacturer's instructions, the tissue sections were incubated overnight at 4 °C in the dark with the unconjugated primary antibody, and then incubated with the fluorescent dye-labeled secondary antibody for three hours at 4 °C in the dark. The primary and secondary antibodies are shown in Table 2. The tissue sections were counterstained with DAPI (Invitrogen) to visualize the cell nuclei. The contribution of transplanted cells to neovascularization was captured by a Zeiss LSM 700 or 780 confocal microscope (Carl Zeiss AG).

[0200] Results

[0201] rSMCs promote microvascular formation through pericyte coating. To investigate the in vivo behavior of transplanted rSMCs, rSMCs were pre-labeled with the red fluorescent dye CM-DiI. DiI-pre-labeled rSMCs were transplanted into three sites of the ischemic hindlimb. Twenty-eight days after transplantation, fluorescein-conjugated ILB4 was systemically injected to identify functional endothelium( Figure 67 ). The distribution of transplanted rSMCs relative to ILB4+ vessels at various levels of the vascular tree was determined. As Figure 68 shown, cross-sections of the ischemic hindlimb muscle injected with cells were visualized. This view shows that a large number of transplanted rSMCs (arrows) are located near capillariesized vessels (diameter < approximately 10.0 μm)( Figure 69 ). These findings imply that rSMCs can promote angiogenesis through pericyte coating. As Figure 70 shown, longitudinal sections of the ischemic hindlimb muscle injected with cells were also visualized. In the longitudinal sections, rSMCs (arrows) are located in vessels with a diameter > 20 μm( Figure 71 ).

[0202] rSMCs promote microvessel formation as vascular SMCs. ACTA2 immunostaining further showed that a subset of rSMCs expressing ACTA2 (ACTA2+ DiI+, arrows) formed a narrow circumferential band around blood vessels with a diameter of approximately 25 μm (curved dashed line)( Figure 72 ). These findings imply that rSMCs can contribute to the smooth muscle cell layer of larger microvessels. To verify the identity of rSMCs as vascular SMCs in vivo, immunostaining for SMTN, a marker of mature contractile SMCs, was performed (see, e.g., Owens et al., Physiological Rev., 84:767 - 801 (2004) and van der Loop et al., Arterioscler. Thomb. Vasc. Biol., 17:665 - 671 (1997)). Confocal microscopy showed that DiI+ rSMCs expressed SMTN and were located in the vessel wall of vessels with a diameter of approximately 10 to 35 μm( Figures 73 - 74 / 77 - 78). Some rSMCs clearly contributed to the smooth muscle layer of the wall, indicating their contribution as vascular SMCs. Since the transplanted rSMCs were heterologous, consisting of a mixed population of HDFs and rSMCs, the fate of DiI+ HDFs similarly injected into the hindlimb muscle was examined to compare the results obtained above( Figures 75 - 76 ). Confocal microscopy showed that HDFs rarely localized to capillariesized vessels and were rarely present at relatively larger vessels stained for ACTA2( Figures 75 - 76 ). These findings suggest that HDFs appear to have a minimal contribution to angiogenesis. Collectively, these findings indicate that transplanted rSMCs can promote angiogenesis through pericytes in capillariesized vessels and vascular SMCs in larger vessels.

[0203] Example 6: Effect of rSMCs on Vascular Permeability

[0204] Materials and methods

[0205] In vitro vascular permeability assay. Cells were seeded at a density of 2.0 x 10 5 cells / ml on the outer side of the wells of 0.4 μm pore size 24 - well inserts (Corning) and incubated at 37 °C and 5% CO2 for four hours. Human umbilical vein ECs (HUVECs) were seeded at 1.0 x 10 5Cells were seeded at a density of cells / ml on the luminal side of the insert and incubated overnight at 37 °C and 5% CO2. The insert was transferred to fresh EGM-2 medium and carefully replaced with EGM-2 medium containing 1 mg / ml FITC-dextran (Sigma-Aldrich), and then incubated for four hours at 37 °C and 5% CO2. Cells were harvested, and the intensity of FITC-dextran was measured by a Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific), with excitation and emission of green fluorescence at 485 and 535 nm, respectively.

[0206] Results

[0207] rSMCs can restrict vascular permeability. The wrapping of the vessel wall by mural cells tightly regulates vascular permeability, thereby restricting extravasation (see, e.g., Armulik et al., Dev. Cell, 21:193-215 (2011); Armulik et al., Nature, 468:557-561 (2010)). To determine whether rSMCs can regulate vascular permeability, an in vitro co-culture model was employed, in which HUVECs and rSMCs were seeded on opposite sides of a semi-permeable membrane ( Figure 79 ). Then, FITC-dextran diffused through the membrane over time, and the fluorescence intensity of dextran in the lower chamber was measured ( Figure 80 ). The identities of HUVECs and rSMCs were verified by immunostaining for PECAM1 and CNN1, respectively ( Figure 81 ). Quantitatively, when HUVECs were co-cultured with rSMCs, the total fluorescence (permeability) of FITC-dextran was significantly lower than that in monoculture or co-culture with HDFs. The results indicate that the presence of rSMCs forms a barrier that controls vascular permeability.

[0208] Example 7: Paracrine effect of rSMCs on ischemia

[0209] Methods

[0210] Hindlimb ischemia, cell transplantation, and qRT-PCR. Induction of hindlimb ischemia and cell transplantation were performed as described above. qRT-PCR was performed as previously described, using the primers described in Table 6.

[0211] Table 6. Primers used for qRT-PCR analysis in Example 7

[0212]

[0213] Results

[0214] Increased angiogenic gene expression in HLI muscles treated with rSMC. To gain insight into the potential mechanisms underlying the early therapeutic effects of rSMC on hindlimb ischemia, qRT-PCR was performed on muscles harvested one week after surgery (Figure 82). The expression of three representative angiogenic genes, Angpt1, Fgf2, and Vegfa, was determined (see, e.g., Carmeliet, Nat. Med., 6:389-395 (2000)). The mRNA expression of Angpt1 and Fgf2 was significantly increased in rSMC-HLI muscles compared to HDF-treated HLI muscles and untreated HLI muscles. Although the expression of Vegfa was much higher in the HDF group than in the rSMC group, the expression of Hif1a, an upstream regulator of Vegfa, was higher in the rSMC group. These results suggest that both HDF and rSMC can exert paracrine effects to prevent ischemic injury.

[0215] Increased angiogenic gene expression in HLI muscles treated with rSMC. Next, as shown in Fig. 82, the expression of eight representative angiogenic genes, Ccl2, its receptor Ccr2, Tgfb1, Pdgfb, Csf1, and Mmps (2, 3, and 9), was determined (see, e.g., Cai et al., Am. J. Heart Circ. Physiol., 284: H31-40 (2003) and Heil & Schaper, Circ. Res., 95: 449-458 (2004)). Except for Tgfb1, the mRNA expression of these angiogenic genes was higher in rSMC-HLI muscles than in HDF-treated HLI muscles and untreated HLI muscles (Fig. 82). Additionally, as shown in Fig. 82, the expression of two members of the Notch signaling pathway (Hey1 and Dll4) and Igf1 and Hgf was examined. Two members of the Notch signaling pathway, Hey1 and Dll4, play important roles in vascular stabilization and maturation (see, e.g., Hoglund & Majesky, Circulation, 125: 212-215 (2012); Manderfield et al., Circulation, 125: 314-323 (2012); and Scheppke et al., Blood, 119: 2149-2158 (2012)), while Igf1 and Hgf play key roles in improving SMC implantation and vascular function (see, e.g., Liu et al., Am. J. Physiol. Heart Circ. Physiol., 287: H2840-2849 (2004) and Powell et al., Circulation, 118: 58-65 (2008)). The expression levels of all four genes were significantly increased in the rSMC group compared to the HDF group (Fig. 82). These findings suggest that rSMC transplantation not only increases angiogenesis in ischemic limbs but also increases angiogenic and vascular stabilizing factors.

Claims

1. A method for generating reprogrammed smooth muscle cells, the method comprising culturing the fibroblasts together with all - trans retinoic acid (ATRA) under conditions for generating reprogrammed smooth muscle cells from the fibroblasts, wherein the fibroblasts are genetically modified to overexpress myocardin.

2. The method according to claim 1, wherein the conditions for generating the reprogrammed smooth muscle cells from the fibroblasts include contacting the fibroblasts with the ATRA for at least two days.

3. The method according to claim 2, wherein the contacting step lasts for 4 - 8 days.

4. The method according to claim 2 or 3, wherein the contacting step comprises contacting the fibroblasts with ATRA.

5. The method according to any one of claims 1 - 4, wherein the fibroblasts are mammalian fibroblasts.

6. The method according to claim 5, wherein the fibroblasts are human fibroblasts.

7. The method according to claim 6, wherein the human fibroblasts are human dermal fibroblasts.

8. The method according to any one of claims 1 - 7, further comprising genetically modifying the fibroblasts by introducing a heterologous nucleic acid encoding myocardin into the fibroblasts.

9. The method according to claim 8, wherein the heterologous nucleic acid is introduced into the fibroblasts by viral transduction.

10. The method according to claim 8, wherein the heterologous nucleic acid is stably integrated into the fibroblast genome.

11. The method according to claim 8, wherein the heterologous nucleic acid is introduced into the fibroblasts by gene editing.

12. A reprogrammed smooth muscle cell prepared by the method according to any one of claims 1 - 11.

13. A reprogrammed smooth muscle cell comprising a heterologous nucleic acid encoding myocardin, wherein the reprogrammed smooth muscle cell co - expresses CNN1 and SMTN in a non - striated pattern.

14. The reprogrammed smooth muscle cell according to claim 12 or 13, wherein the reprogrammed smooth muscle cell contracts by more than 10% in the presence of carbachol.

15. A composition comprising a population of reprogrammed smooth muscle cells according to any one of claims 12 - 14 and a pharmaceutically acceptable carrier.

16. A method for treating a subject suffering from or at risk of developing ischemia, the method comprising administering to the subject an effective amount of the composition according to claim 15.

17. The method according to claim 16, wherein the effective amount of the composition increases the vascular perfusion of the subject.

18. The method according to claim 16, wherein the effective amount of the composition increases the neovascularization of the subject.

19. The method according to claim 16, wherein the effective amount of the composition increases the arteriogenesis of the subject.

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