Gina D. Kusuma

Decidua Parietalis-Derived Mesenchymal Stromal Cells Reside in a Vascular Niche Within the Choriodecidua

Mesenchymal stromal cells (MSCs) from gestational tissues represent promising cell populations with stem cell-like properties for use in regenerative medicine. Previously, we reported that MSCs in the chorionic villi of the human placenta reside in a vascular niche. However, the niche(s) in which MSCs reside in the fetal membranes, another rich source of MSCs, remains to be determined. The cell surface markers STRO-1 and 3G5 were previously employed to identify niches in a variety of tissues and here we use these markers to report the location of the MSC niche in the human decidua parietalis. The cultured decidua parietalis MSCs (DPMSCs) isolated from the choriodecidua component of the fetal membranes possessed stem cell-like properties such as adherence to plastic, colony forming ability, and multipotent differentiation potential. Fluorescence in situ hybridization analysis showed cultured DPMSCs were of maternal origin. Immunocytochemistry demonstrated that cultured DPMSCs stained positively with stem cell surface markers 3G5, CD105, CD106, STRO-1, CD146, CD49a, and α-SMA but were negative for hematopoietic markers (CD117, CD34) and vascular markers (CD34, von Willebrand factor [vWF]). Immunohistochemistry with antibodies to stem cell surface markers and the endothelial markers on term fetal membranes revealed a vascular niche for DPMSCs, which was confirmed by immunofluorescence analysis. Both STRO-1 and vWF fluorescence signals showed substantial overlap, while CD146 and vWF signals showed partial overlap. These observations were consistent with a vascular niche.

Mesenchymal Stem/Stromal Cells Derived From a Reproductive Tissue Niche Under Oxidative Stress Have High Aldehyde Dehydrogenase Activity

The use of mesenchymal stem/stromal cells (MSC) in regenerative medicine often requires MSC to function in environments of high oxidative stress. Human pregnancy is a condition where the mother’s tissues, and in particular her circulatory system, are exposed to increased levels of oxidative stress. MSC in the maternal decidua basalis (DMSC) are in a vascular niche, and thus would be exposed to oxidative stress products in the maternal circulation. Aldehyde dehydrogenases (ALDH) are a large family of enzymes which detoxify aldehydes and thereby protect stem cells against oxidative damage. A subpopulation of MSC express high levels of ALDH (ALDHbr) and these are more potent in repairing and regenerating tissues. DMSC was compared with chorionic villous MSC (CMSC) derived from the human placenta. CMSC reside in vascular niche and are exposed to the fetal circulation, which is in lower oxidative state. We screened an ALDH isozyme cDNA array and determined that relative to CMSC, DMSC expressed high levels of ALDH1 family members, predominantly ALDH1A1. Immunocytochemistry gave qualitative confirmation at the protein level. Immunofluorescence detected ALDH1 immunoreactivity in the DMSC and CMSC vascular niche. The percentage of ALDHbr cells was calculated by Aldefluor assay and DMSC showed a significantly higher percentage of ALDHbr cells than CMSC. Finally, flow sorted ALDHbr cells were functionally potent in colony forming unit assays. DMSC, which are derived from pregnancy tissues that are naturally exposed to high levels of oxidative stress, may be better candidates for regenerative therapies where MSC must function in high oxidative stress environments.

Reduced aldehyde dehydrogenase expression in preeclamptic decidual mesenchymal stem/stromal cells is restored by aldehyde dehydrogenase agonists

High resistance to oxidative stress is a common feature of mesenchymal stem/stromal cells (MSC) and is associated with higher cell survival and ability to respond to oxidative damage. Aldehyde dehydrogenase (ALDH) activity is a candidate “universal” marker for stem cells. ALDH expression was significantly lower in decidual MSC (DMSC) isolated from preeclamptic (PE) patients. ALDH gene knockdown by siRNA transfection was performed to create a cell culture model of the reduced ALDH expression detected in PE-DMSC. We showed that ALDH activity in DMSC is associated with resistance to hydrogen peroxide (H2O2)-induced toxicity. Our data provide evidence that ALDH expression in DMSC is required for cellular resistance to oxidative stress. Furthermore, candidate ALDH activators were screened and two of the compounds were effective in upregulating ALDH expression. This study provides a proof-of-principle that the restoration of ALDH activity in diseased MSC is a rational basis for a therapeutic strategy to improve MSC resistance to cytotoxic damage.

Ectopic Bone Formation by Mesenchymal Stem Cells Derived from Human Term Placenta and the Decidua.

Mesenchymal stem cells (MSCs) are one of the most attractive cell types for cell-based bone tissue repair applications. Fetal-derived MSCs and maternal-derived MSCs have been isolated from chorionic villi of human term placenta and the decidua basalis attached to the placenta following delivery, respectively. Chorionic-derived MSCs (CMSCs) and decidua-derived MSCs (DMSCs) generated in this study met the MSCs criteria set by International Society of Cellular Therapy. These criteria include: (i) adherence to plastic; (ii) >90% expression of CD73, CD105, CD90, CD146, CD44 and CD166 combined with <5% expression of CD45, CD19 and HLA-DR; and (iii) ability to differentiate into osteogenic, adipogenic, and chondrogenic lineages. In vivo subcutaneous implantation into SCID mice showed that both bromo-deoxyuridine (BrdU)-labelled CMSCs and DMSCs when implanted together with hydroxyapatite/tricalcium phosphate particles were capable of forming ectopic bone at 8-weeks post-transplantation. Histological assessment showed expression of bone markers, osteopontin (OPN), osteocalcin (OCN), biglycan (BGN), bone sialoprotein (BSP), and also a marker of vasculature, alpha-smooth muscle actin (α-SMA). This study provides evidence to support CMSCs and DMSCs as cellular candidates with potent bone forming capacity.

A Novel Combination of Homeobox Genes is Expressed in Mesenchymal Chorionic Stem/Stromal Cells in First Trimester and Term Pregnancies

Human chorionic mesenchymal stem/stromal cells (CMSCs) derived from the placenta are similar to adult tissue-derived MSCs. The aim of this study was to investigate the role of these cells in normal placental development. Transcription factors, particularly members of the homeobox gene family, play crucial roles in maintaining stem cell proliferation and lineage specification in embryonic tissues. In adult tissues and organs, stem cells proliferate at low levels in their niche until they receive cues from the microenvironment to differentiate. The homeobox genes that are expressed in the CMSC niche in placental tissues have not been identified. We used the novel strategy of laser capture microdissection to isolate the stromal component of first trimester villi and excluded the cytotrophoblast and syncytiotrophoblast layers that comprise the outer layer of the chorionic villi. Microarray analysis was then used to screen for homeobox genes in the microdissected tissue. Candidate homeobox genes were selected for further RNA analysis. Immunohistochemistry of candidate genes in first trimester placental villous stromal tissue revealed homeobox genes Meis1, myeloid ectropic viral integration site 1 homolog 2 (MEIS2), H2.0-like Drosophila (HLX), transforming growth factor β-induced factor (TGIF), and distal-less homeobox 5 (DLX5) were expressed in the vascular niche where CMSCs have been shown to reside. Expression of MEIS2, HLX, TGIF, and DLX5 was also detected in scattered stromal cells. Real-time polymerase chain reaction and immunocytochemistry verified expression of MEIS2, HLX, TGIF, and DLX5 homeobox genes in first trimester and term CMSCs. These data suggest a combination of regulatory homeobox genes is expressed in CMSCs from early placental development to term, which may be required for stem cell proliferation and differentiation.

Decellularized extracellular matrices produced from immortal cell lines derived from different parts of the placenta support primary mesenchymal stem cell expansion

Mesenchymal stem/stromal cells (MSCs) exhibit undesired phenotypic changes during ex vivo expansion, limiting production of the large quantities of high quality primary MSCs needed for both basic research and cell therapies. Primary MSCs retain many desired MSC properties including proliferative capacity and differentiation potential when expanded on decellularized extracellular matrix (dECM) prepared from primary MSCs. However, the need to use low passage number primary MSCs (passage 3 or lower) to produce the dECM drastically limits the utility and impact of this technology. Here, we report that primary MSCs expanded on dECM prepared from high passage number (passage 25) human telomerase reverse transcriptase (hTERT) transduced immortal MSC cell lines also exhibit increased proliferation and osteogenic differentiation. Two hTERT-transduced placenta-derived MSC cell lines, CMSC29 and DMSC23 [derived from placental chorionic villi (CMSCs) and decidua basalis (DMSCs), respectively], were used to prepare dECM-coated substrates. These dECM substrates showed structural and biochemical differences. Primary DMSCs cultured on dECM-DMSC23 showed a three-fold increase in cell number after 14 days expansion in culture and increased osteogenic differentiation compared with controls. Primary CMSCs cultured on the dECM-DMSC23 exhibited a two-fold increase in cell number and increased osteogenic differentiation. We conclude that immortal MSC cell lines derived from different parts of the placenta produce dECM with varying abilities for supporting increased primary MSC expansion while maintaining important primary MSC properties. Additionally, this is the first demonstration of using high passage number cells to produce dECM that can promote primary MSC expansion, and this advancement greatly increases the feasibility and applicability of dECM-based technologies.

Isolation and Characterization of Mesenchymal Stem/Stromal Cells Derived from Human Third Trimester Placental Chorionic Villi and Decidua Basalis

The decidua basalis and placental chorionic villi are critical components of maternal-fetal interface, which plays a critical role in normal placental development. Failure to form a proper maternal-fetal interface is associated with clinically important placental pathologies including preeclampsia and fetal growth restriction. Placental trophoblast cells are well known for their critical roles in establishing the maternal-fetal interface; however accumulating evidence also implicates mesenchymal stem/stromal cells that envelop the maternal and fetal blood vessels as playing an important role in the formation and efficient functioning of the interface. Moreover, recent studies associate abnormal mesenchymal stem/stromal cell function in the development of preeclampsia. Further research is needed to fully understand the role that these cells play in this clinically important placental pathology.The intimate relationship between maternal and fetal tissues at the interface poses significant problems in the enrichment of decidua basalis and chorionic villous mesenchymal stem/stromal cells without significant cross-contamination. The protocols described below for the enrichment and characterization of mesenchymal stem/stromal cells from the maternal-fetal interface produce highly enriched cells that conform to international standards and show minimal cross-contamination.