Fig 1.
Identification of CD133+ cells in LMs of different subtypes and anatomical locations.
(A) LYVE1 and podoplanin staining of cervicofacial mixed LM tissue and patient-matched uninvolved tissue. White arrowheads mark normal lymphatics. (B) Podoplanin and CD133 staining of neonatal foreskin (postnatal day 1), uninvolved tissue, mixed cervicofacial (Mixed CF) LM tissues (2x), and Gorham’s dermal tissue. White arrowheads mark CD133+/podoplanin+ lymphatic endothelium. Red arrowheads mark CD133low/podoplanin+ lymphatic endothelium. Yellow arrowheads mark CD133+/podoplanin+ stromal cells. Blue asterisks mark blood vessels with autofluorescing red blood cells. Scale bars: 50μm. lymphatic channel (lc)
Fig 2.
Endothelial precursor and lymphatic endothelial protein expression in isolated CD133+ and CD133− LM cells.
FACS of patient-matched CD133+ and CD133− LM cells isolated from microcystic mesenteric (Micro Mes) LM, microcystic subcutaneous (Micro SC) LM and general lymphatic anomaly (GLA) specimens. (A) Endothelial precursor markers, CD34, CD90, CD146 and VEGFR-2. (B) Lymphatic endothelial cell markers, podoplanin and VEGFR-3. Blue line represents antibody data, and red line IgG control.
Fig 3.
Expression of markers for mature lymphatic endothelial cells in isolated CD133+ and CD133− LM cells.
(A) Podoplanin, VE-cadherin, VEGFR-2, VEGFR-3, Prox1, and LYVE1 qRT-PCR of RNA isolated from CD133+ and CD133− cells from LMs of different subtypes and anatomical locations and GLA compared to control HdLECs. Data normalized to β-actin qRT-PCR and represented as mean ± s.e.m. (B) CD31 and VE-cadherin FACS of patient-matched CD133+ and CD133− LM cells isolated from microcystic mesenteric (Micro Mes) LM, microcystic subcutaneous (Micro SC) LM and general lymphatic anomaly (GLA) specimens. Thick gray line represents antibody data, and black line IgG control. (C) VE-cadherin/CD31 and (D) podoplanin/LYVE1 staining of patient-matched CD133+ and CD133− LM cells isolated from microcystic mesenteric (Micro Mes) LM, microcystic subcutaneous (Micro SC) LM, and GLA compared to control HdLEC. Scale bars: 50μm.
Fig 4.
Differentiation of LMPCs into fat, bone and smooth muscle cells.
(A) Oil Red O staining of LMPCs isolated from macrocystic cervicofacial (Macro CF) LM and microcystic mesenteric (Micro Mes) LM after 2 weeks in growth media (control) or adipogenic media. (B) Alkaline phosphatase (Alk Phos) staining of LMPCs isolated from macrocystic Macro CF LM and Micro Mes LM after 2 weeks in growth media (control) or osteogenic media. (C) NG2 and alpha smooth muscle actin (αSMA) staining of LMPCs isolated from Micro Mes LM after 2 weeks in growth media (control) or mural cell differentiation (Diff) media. Scale bars: 50μm.
Fig 5.
LMPCs differentiated into abnormal lymphatic endothelial cells.
LMPCs isolated from mixed cervicofacial (Mixed CF) LM, macrocystic mesenteric (Macro Mes) LM and a generalized lymphatic anomaly (GLA) were maintained in growth media (control) or lymphatic endothelial differentiation (LEC Diff) media for two weeks. (A) CD31/VE-cadherin and (B) LYVE1/podoplanin staining. Scale bars: 50μm. (C) VEGFR-1, VEGFR-2, VE-cadherin, Prox1, Podoplanin, LYVE1, and VEGFR-3 qRT-PCR of RNA isolated from LMPCs maintained in growth or LEC differentiation (LEC Diff) media. Data normalized to β-actin qRT-PCR and represented as mean ± s.e.m. * p < 0.05, ** p < 0.005, *** p < 0.0005.
Fig 6.
LMPC recapitulated the LM phenotype in a mouse model.
LMPCs isolated from mixed cervicofacial (Mixed CF), macrocystic cervicofacial (Macro CF) and mixed mesenteric (Mixed Mes) LM were suspended in Matrigel and implanted into GFP-expressing immunocompromised mice. Matrigel alone, mesenchymal stem cells (MSCs), normal human dermal lymphatic endothelial cells (HdLEC) and hemangioma stem cells (HemSC) served as controls. (A) Hematoxylin and eosin (H&E) staining of xenograft sections. (B) GFP (host cell) and podoplanin staining of xenograft sections. Scale bars: 50μm. lymphatic channel (lc)
Fig 7.
Patient-matched LMPC and LMEC implants expressed the lymphatic proteins, LYVE1 and VEGFR-3.
CD133+ LMPCs and CD133- LMECs isolated from a microcystic subcutaneous LM were suspended in Matrigel and implanted in immunocompromised mice. Staining of implants was compared to microcystic subcutaneous LM patient tissue (Micro LM tissue). (A) Podoplanin and LYVE1 and (B) podoplanin and VEGFR-3 staining. Scale bars: 50μm.
Fig 8.
Patient-matched LMPC and LMEC implants expressed the lymphatic endothelial master regulator, Prox1.
CD133+ LMPCs and CD133− LMECs isolated from a microcystic subcutaneous LM were suspended in Matrigel and implanted in immunocompromised mice. Podoplanin and Prox1 staining of implants was compared to microcystic subcutaneous LM patient tissue (Micro LM tissues). Arrows mark Prox1 positive nuclei. Boxed areas enlarged below. Scale bars: 50μm.