Differentiation of Human Amniotic Mesenchymal Stem Cells into Human Anterior Cruciate Ligament Fibroblast Cells by In Vitro Coculture

Abstract

Anterior cruciate ligament injuries are common in humans, though cellular components of the knee have little regenerative or proliferation potential. This study investigated the differentiation of human amnion-derived mesenchymal stem cells (hAMSCs) into human anterior cruciate ligament fibroblasts (hACLFs) in vitro through induction with bFGF and TGF-β1 with coculture systems. Groups A and B comprised hAMSCs at the 3rd passage cultured with and without bFGF and TGF-β1, respectively; Groups C and D consisted of hAMSCs and hACLFs in monolayer coculture with and without bFGF and TGF-β1, respectively; Groups E and F were composed of hAMSCs and hACLFs in Transwell coculture with and without bFGF and TGF-β1, respectively. Cell morphology and proliferation were recorded. Protein expression and relative mRNA expression were evaluated in each group. Cell proliferation was significantly higher in the induced groups than in the noninduced groups. Protein expression increased over time with the highest expression observed in Group E. mRNA levels were significantly higher in Group E than in other groups. This study is the first to demonstrate the use of the Transwell coculture system for this purpose, and hAMSCs were successfully differentiated into hACLFs. Thus, hAMSCs may be a superior choice for hACLF differentiation via Transwell coculture.

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“…hAMSCs were isolated from six full-term puerperants, and informed consent was obtained from all of the patients prior to their participation. For the isolation of hAMSCs, they were first dissected bluntly from the placenta as previously described 13 . Following this, the amnion samples were washed three times with phosphate-buffered saline (PBS) and transferred to sterile containers at 4°C in a laboratory facility.…”

“…In addition, they do not carry ethical or moral controversies with them 12 . hAMSCs have a multidirectional differentiation capacity as well as an advantage in therapeutic angiogenesis due to the hAMSC root in placenta, which is a vascular tissue 13 . Hence, hAMSCs have been extensively applied in the treatment of bone and spinal traumas and vascular reconstruction surgery.…”

FGF-2-Induced Human Amniotic Mesenchymal Stem Cells Seeded on a Human Acellular Amniotic Membrane Scaffold Accelerated Tendon-to-Bone Healing in a Rabbit Extra-Articular Model

“…The results showed that hAMSCs expressed vimentin-a marker for MSCs-greatly but hardly expressed cytokeratin 19 (a typical phenotype molecule of human amniotic epithelial cells). Although vimentin was used as the only hAMSC marker, other properties of hAMSCs were confirmed in our previous experiment [35], such as plastic adherence, specific surface antigen (Ag) expression, and multipotent ability to differentiate into osteoblasts, adipocytes, and chondrocytes. After confirming MSC properties in our P3 hAMSC culture lines, we used a lentivirus containing the human FGF-2 gene to transfect the hAMSCs wherein the transfection efficiency was determined via the expression of fluorescence and qRT-PCR.…”

“…For this study, placentas were obtained from the Obstetrics Department of the Affiliated Hospital of Zunyi Medical University with informed consent from each patient before operation. In line with a previous research [35], hAMSCs used in this study were isolated from the human placental amniotic membrane of 5 healthy full-term puerperant women by subjecting to enzymatic (Solarbio, China) digestion twice, followed by collagenase type II (Gibco, USA) digestion once. In brief, the isolated amniotic membrane was minced into pieces and digested twice with 0.05% trypsin/0.01% ethylenediaminetetraacetic acid (EDTA) for 30 mins at 37°C, followed by washing the tissue with phosphate buffer solution (PBS) and digestion with 0.75% collagenase type II for 1 h at 37°C.…”

“…Third-generation (P3) hAMSCs of good condition obtained by this method were used for subsequent experiments. According to the established protocol [35], the stemness of the hAMSCs isolated from placental amniotic membrane was tested via immunostaining with a stem cell marker and a human amniotic epithelial cell marker, vimentin (1 : 300, ab92547, Abcam) and cytokeratin 19 (CK-19, 1 : 200, ab52625, Abcam), respectively.…”