ABSTRACT
Current protocols for the differentiation of human pluripotent stem cells (hPSCs) into chondrocytes do not allow for the expansion of intermediate progenitors so as to prospectively assess their chondrogenic potential. Here we report a protocol that leverages PRRX1-tdTomato reporter hPSCs for the selective induction of expandable and ontogenetically defined PRRX1+ limb-bud-like mesenchymal cells under defined xeno-free conditions, and the prospective assessment of the cells' chondrogenic potential via the cell-surface markers CD90, CD140B and CD82. The cells, which proliferated stably and exhibited the potential to undergo chondrogenic differentiation, formed hyaline cartilaginous-like tissue commensurate to their PRRX1-expression levels. Moreover, we show that limb-bud-like mesenchymal cells derived from patient-derived induced hPSCs can be used to identify therapeutic candidates for type II collagenopathy and we developed a method to generate uniformly sized hyaline cartilaginous-like particles by plating the cells on culture dishes coated with spots of a zwitterionic polymer. PRRX1+ limb-bud-like mesenchymal cells could facilitate the mass production of chondrocytes and cartilaginous tissues for applications in drug screening and tissue engineering.
Subject(s)
Homeodomain Proteins/genetics , Mesenchymal Stem Cells/metabolism , Pluripotent Stem Cells/cytology , Animals , Cell Culture Techniques/methods , Cell Differentiation , Chondrocytes/cytology , Chondrocytes/metabolism , Chondrocytes/transplantation , Chondrogenesis , Collagen Diseases/therapy , Culture Media/chemistry , Homeodomain Proteins/metabolism , Humans , Mesenchymal Stem Cells/cytology , Mice , Mice, Inbred NOD , Mice, SCID , Pluripotent Stem Cells/metabolism , Polymers/chemistry , Receptor, Platelet-Derived Growth Factor beta/metabolism , Thy-1 Antigens/metabolism , Tissue EngineeringABSTRACT
We started our home hemodialysis (HHD) program in July 2005 and have been promoting overnight HHD. As more than 6 years have passed since we started our HHD program, we review our HHD program and 8 overnight HHD patients (5 males and 3 females). Their underlying disease differs in each and none have diabetic nephropathy. Their average age was 49.2 ± 6.0 years (mean ± SD). Average duration of dialysis treatment, HHD, and overnight HHD was 9.4 ± 4.4, 3.5 ± 2.4, and 2.2 ± 1.7 years, respectively. Average treatment time per dialysis session was 6.9 ± 0.8 h/treatment, average treatment days weekly was 4.5 ± 0.8 days/week, and average treatment time weekly was 31.2 ± 7.0 h/week. Laboratory data were good and their blood pressure was well controlled without any antihypertensive drugs excluding a patient who was recently introduced to dialysis with some residual kidney function. Severe problems did not occur in these 6 years except for blood access infection twice, slipping out of a needle during dialysis with small blood loss once, and a drop in blood pressure at the end of dialysis once, which was recovered by her assistant's help. According to our HHD training program, the average training duration for HHD was 106 ± 42 days. The shortest was 60 days and longest 198 days. These differences among training durations might be because of the frequency of training and having a better hand of puncturing. We did not instruct any additional issues and points for overnight HHD, because performing overnight HHD is similar to standard HHD. Some patients moved to overnight HHD slowly starting with once weekly and the others started overnight HHD several days after they had started HHD.
