Pelvic Organ Distribution of Mesenchymal Stem Cells Injected Intravenously after Simulated Childbirth Injury in Female Rats

The local route of stem cell administration utilized presently in clinical trials for stress incontinence may not take full advantage of the capabilities of these cells. The goal of this study was to evaluate if intravenously injected mesenchymal stem cells (MSCs) home to pelvic organs after simulated childbirth injury in a rat model. Female rats underwent either vaginal distension (VD) or sham VD. All rats received 2 million GFP-labeled MSCs intravenously 1 hour after injury. Four or 10 days later pelvic organs and muscles were imaged for visualization of GFP-positive cells. Significantly more MSCs home to the urethra, vagina, rectum, and levator ani muscle 4 days after VD than after sham VD. MSCs were present 10 days after injection but GFP intensity had decreased. This study provides basic science evidence that intravenous administration of MSCs could provide an effective route for cell-based therapy to facilitate repair after injury and treat stress incontinence

Pelvic Organ Distribution of Mesenchymal Stem Cells Injected Intravenously after Simulated Childbirth Injury in Female Rats

The local route of stem cell administration utilized presently in clinical trials for stress incontinence may not take full advantage of the capabilities of these cells. The goal of this study was to evaluate if intravenously injected mesenchymal stem cells (MSCs) home to pelvic organs after simulated childbirth injury in a rat model. Female rats underwent either vaginal distension (VD) or sham VD. All rats received 2 million GFP-labeled MSCs intravenously 1 hour after injury. Four or 10 days later pelvic organs and muscles were imaged for visualization of GFP-positive cells. Significantly more MSCs home to the urethra, vagina, rectum, and levator ani muscle 4 days after VD than after sham VD. MSCs were present 10 days after injection but GFP intensity had decreased. This study provides basic science evidence that intravenous administration of MSCs could provide an effective route for cell-based therapy to facilitate repair after injury and treat stress incontinence

Bone Marrow SSEA1+ Cells Support the Myocardium in Cardiac Pressure Overload

<div><p>Rationale</p><p>Stage specific embryonic antigen 1+ (SSEA1+) cells have been described as the most primitive mesenchymal progenitor cell in the bone marrow. Cardiac injury mobilizes SSEA1+ cells into the peripheral blood but their <i>in vivo</i> function has not been characterized.</p> <p>Objective</p><p>We generated animals with chimeric bone marrow to determine the fate and function of bone marrow SSEA1+ cells in response to acute cardiac pressure overload.</p> <p>Methods and Results</p><p>Lethally irradiated mice were transplanted with normal bone marrow where the wild-type SSEA1+ cells were replaced with green fluorescent protein (GFP) SSEA1+ cells. Cardiac injury was induced by trans-aortic constriction (TAC). We identified significant GFP+ cell engraftment into the myocardium after TAC. Bone marrow GFP+ SSEA1 derived cells acquired markers of endothelial lineage, but did not express markers of c-kit+ cardiac progenitor cells. The function of bone marrow SSEA1+ cells after TAC was determined by transplanting lethally irradiated mice with bone marrow depleted of SSEA1+ cells (SSEA1-BM). The cardiac function of SSEA1-BM mice declined at a greater rate after TAC compared to their complete bone marrow transplant counterparts and was associated with decreased bone marrow cell engraftment and greater vessel rarefication in the myocardium.</p> <p>Conclusions</p><p>These results provide evidence for the recruitment of endogenous bone marrow SSEA1+ cells to the myocardium after TAC. We demonstrate that, <i>in vivo</i>, bone marrow SSEA1+ cells have the differentiation potential to acquire endothelial lineage markers. We also show that bone marrow SSEA1+ deficiency is associated with a reduced compensatory capacity to cardiac pressure overload, suggesting their importance in cardiac homeostasis. These data demonstrate that bone marrow SSEA1+ cells are critical for sustaining vascular density and cardiac repair to pressure overload.</p> </div

TAC increases fibrosis and <b>myocyte surface area.</b>

<p>(A) Mason’s trichrome staining for collagen (Magnification 20x). (B) TAC (white bars) significantly increased the presence of fibrosis in the hearts compared to sham (black bars) but no difference was observed based on bone marrow. (C) Four weeks after TAC, mice had increased myocyte hypertrophy compared to sham though no differences were determined between bone marrow. (D) Representative immunofluorescence of transversely sectioned myocytes of TAC and sham groups four weeks after surgery (40x Magnification). Myocyte membranes are outlined by WGA (purple) and nuclei are labeled with DAPI (blue). n = 4-6. * P ≤ 0.05 compared to sham. Abbreviations: TAC, trans-aortic constriction; WGA, wheat germ agglutinin; SSEA1, stage-specific embryonic antigen 1; SSEA1-BM. SSEA1+ cell depleted bone marrow; BM, bone marrow; GFP, green fluorescent protein.</p

Bone Marrow Recruitment to the Heart is Reduced when SSEA1 Cells are Depleted from the Bone Marrow.

<p>(A) Bar graph representing the number of bone marrow cells (GFP+) in the myocardium in sham (black bars) and TAC (white bars) animals. In sham animals, there are significantly fewer bone marrow cells in the heart of SSEA1-BM mice compared to animals with total BM. Four weeks after TAC, GFP+ cells were significantly increased in the myocardium in mice with total BM but not in SSEA1-BM mice. (B) Quantification of the percentage of total vessels that are GFP+ (bone marrow derived) in sham and TAC animals. The percentage significantly increased after TAC in mice with total BM compared to sham. No differences were measured between sham and TAC SSEA1-BM mice or between sham groups. (C) Representative immunoflourescence images of bone marrow recruitment and bone marrow participation in angiogenesis in the myocardium (40x). Bone marrow cells are identified as GFP+ (red), vessels as Isolectin B4 + (green), and nuclei are labeled by DAPI (blue). n = 4-6. # P = 0.006 between sham animals. * P < 0.05 compared to respective sham. Abbreviations: SSEA1, stage-specific embryonic antigen 1; GFP, green fluorescent protein; TAC, trans-aortic constriction; SSEA1-BM, SSEA1+ cell depleted bone marrow; BM, bone marrow.</p

Fate of Bone Marrow SSEA1+ cells after TAC.

<p>(A) Experimental Protocol. SSEA1+ cells were depleted from the bone marrow (grey circles) of a C57 mouse (grey mouse) by FACS and replaced by bone marrow SSEA1+ cells from a GFP transgenic mouse (A1, green circles, green mouse). The chimeric bone marrow was injected into lethally irradiated C57 mice. Bar graphs from flow cytometry analysis of SSEA1+ and GFP+ cells in the bone marrow (B) and heart (D) from sham (black bars) and 7 days post-TAC (white bars). The percentage of GFP+ cells contributing to stem cell populations in the bone marrow (C) and heart (E) in sham and TAC (7 days post) animals was analyzed by flow cytometry. Bone marrow SSEA1+ cells contributed to the HSC and EPC, and SSEA1+ cell populations but not to the CSC population. The percentage of SSEA1+ (black wedge), CSC (pink wedge), EPC (grey wedge), and HSC (red wedge) of the total GFP+ cell pool in the bone marrow (F) and myocardium (G) 7 days post-TAC. n = 3-5 per group. * P < 0.05 compared to sham. # P = 0.05 compared to SSEA1+ cells. Abbreviations: SSEA1, stage-specific embryonic antigen 1; TAC, trans-aortic constriction; FACS, fluorescence-activated cell sorting; GFP, green fluorescent protein; HSC, hematopoietic stem cells; EPC, endothelial progenitor cells; CSC, cardiac stem cells.</p

Bone Marrow SSEA1+ Cell Depletion Accelerates Cardiac Remodeling after TAC.

<p>(A) Experimental Protocol. SSEA1+ cells (green circles with red outline) were depleted from the total bone marrow of GFP transgenic mice by FACS and transplanted into lethally irradiated C57 mice (grey; SSEA1-BM). Control mice were transplanted with total GFP bone marrow (total BM). (B) Four weeks after TAC, the heart weight (HW/BW) was increased in both SSEA1-BM (black bars) and total BM (white bars) groups. (C) After TAC, the spleen weight (SW/BW) in SSEA1-BM mice was significantly lower compared to mice with total BM. (D) Measurement of lung weight (LW/BW) 4 weeks post-TAC. (E) The ejection fraction of SSEA1-BM mice (black circle) in response to TAC was decreased quicker relative to mice with total BM (black square). (F) The cardiac function of SSEA1-BM sham mice (open circle) decreased over time. Cardiac function of mice with total BM (open square) remained unchanged. n = 4-6. * P < 0.05 compared to SSEA1-BM sham. # P ≤ 0.01 compared to total BM Sham. ¢ P = 0.05 SSEA1-BM TAC versus total BM TAC. $ P = 0.04 SSEA1-BM Sham versus total BM Sham. Abbreviation: SSEA1, stage-specific embryonic antigen 1; TAC, trans-aortic constriction; GFP, green fluorescent protein; FACS, fluorescence-activated cell sorting; SSEA1-BM, SSEA1+ depleted bone marrow; BM, bone marrow; HW, heart weight; BW, body weight; SW, spleen weight; LW, lung weight.</p

Depletion of SSEA1 from the Bone Marrow Increases Vessel Rarefaction after TAC.

<p>(A) Representative images used to quantify vessel density (Magnification 40x). Vessels were identified as Isolectin+ (Red) and nuclei are labeled with DAPI (blue). Representative images of GFP+ bone marrow derived (Green) alone and their co-localization with the vasculature (Isolectin, Red). Vessel density was quantified as Isolectin+/mm². (B) Mice with SSEA1-BM had significantly decreased vessel density 28 days after TAC compared to sham (P = 0.03). No significant change was measured in TAC mice with total BM (P = 0.28). No differences were identified between sham animals. n = 4-6 per group. (C) Data representing the number of bone marrow derived cells (GFP+) in the myocardium in sham (black bars) and TAC (white bars) animals. In sham animals, there are significantly fewer bone marrow cells in the heart of SSEA1 depleted BM mice compared to animals with total BM. Four weeks after TAC, GFP+ cells were significantly increased in the myocardium in mice with total BM but not in SSEA1-BM mice. (D) Quantification of the percentage of total vessels that are GFP+ (bone marrow derived) in sham and TAC animals. The percentage significantly increased after TAC in mice with total BM compared to sham. No differences were measured between sham and TAC SSEA1 depleted BM mice or between sham groups. Abbreviations: SSEA1, stage-specific embryonic antigen 1; TAC, trans-aortic constriction; SSEA1 depleted BM, SSEA1+ cell depleted bone marrow; BM, bone marrow.</p