Arijit Biswas

Human Wharton's Jelly Stem Cells Have Unique Transcriptome Profiles Compared to Human Embryonic Stem Cells and Other Mesenchymal Stem Cells

The human umbilical cord that originates from the embryo is an extra-embryonic membrane and the Wharton’s jelly within it is a rich source of stem cells (hWJSCs). It is not definitely known whether these cells behave as human embryonic stem cells (hESCs), human mesenchymal stem cells (hMSC) or both. They have the unique properties of high proliferation rates, wide multipotency, hypoimmunogenicity, do not induce teratomas and have anticancer properties. These advantages are important considerations for their use in cell based therapies and treatment of cancers. In a search for properties that confer these advantages we compared a detailed transcriptome profiling of hWJSCs using DNA microarrays with that of a panel of known hESCs, hMSCs and stromal cells. hWJSCs expressed low levels of the pluripotent embryonic stem cell markers including POUF1, NANOG, SOX2 and LIN28, thus explaining why they do not produce teratomas. Several cytokines were significantly upregulated in hWJSCs including IL12A which is associated with the induction of apoptosis, thus explaining their anticancer properties. When GO Biological Process analysis was compared between the various stem cell types, hWJSCs showed an increased expression of genes associated with the immune system, chemotaxis and cell death. The ability to modulate immune responses makes hWJSCs an important compatible stem cell source for transplantation therapy in allogeneic settings without immunorejection. The data in the present study which is the first detailed report on hWJSC transcriptomes provide a foundation for future functional studies where the exact mechanisms of these unique properties of hWJSCs can be confirmed.

Comparative growth behaviour and characterization of stem cells from human Wharton's jelly

Human embryonic stem cells (hESC) face ethical sensitivities and the problem of teratoma formation. Although Whartons jelly stem cells (WJSC), also of embryonic origin, may not face such ethical concerns, it is not definitely known whether under hESC culture conditions they would be as pluripotent as hESC. WJSC grown on plastic showed two types of morphology (epithelioid and short fibroblastic) in primary culture depending on the culture medium used, and only fibroblastic morphology when passaged. When grown in the presence of hESC medium on mouse feeder cells, they produced atypical colonies containing hESC-like cells with high-nuclear cytoplasmic ratios and prominent nucleoli. They were positive for the hESC markers Tra-1-60, Tra-1-81, SSEA-1, SSEA-4, Oct-4 and alkaline phosphatase, negative for SSEA-3, showed normal karyotypes, developed embryoid body (EB)-like structures, did not produce teratomas in SCID mice and differentiated into neuronal derivatives. They were also positive for the mesenchymal CD markers (CD105, CD90, CD44), negative for CD34 and HLA, and although nine out of 10 embryonic stem cell genomic markers were detectable, these were expressed at low levels. WJSC are thus not as pluripotent as hESC but widely multipotent, and have the advantages of being able to be scaled up easily and not inducing teratomas.

Derivation efficiency, cell proliferation, freeze-thaw survival, stem-cell properties and differentiation of human Wharton's jelly stem cells

Human mesenchymal stem cells (MSC) are non-controversial multipotent stem cells. Their presence in umbilical cord blood (UCB) has been debated in some studies and others report low counts per cord blood unit and poor proliferation rates. On the other hand, Whartons jelly of human umbilical cords appears to be a rich source of human MSC. This study derived 13 human Whartons jelly stem cell (WJSC) lines from 13 human umbilical cords (100%) and recovered 4.7 +/- 0.2 x 10(6) live WJSC/cm of cord before culture. Complex culture medium produced greater proliferation rates of the WJSC in culture compared with simple medium. The mean population doubling times were 24.47 +/- 0.33 to 26.25 +/- 0.50 h in complex medium. The stem-cell markers of the WJSC were retained for at least 10 passages in both media. After programmed machine freezing, the thaw-survival rates of WJSC were 85-90% and they could be differentiated into neurons. Given the high derivation efficiency, availability of large numbers of fresh live cells, high expansion capabilities, prolonged maintenance of stem-cell properties and differentiation potential, it is proposed that human WJSC may be frozen at the same time as UCB in cord blood banks for regenerative medicine purposes.

Pre- and Postnatal Transplantation of Fetal Mesenchymal Stem Cells in Osteogenesis Imperfecta: A Two-Center Experience

Osteogenesis imperfecta (OI) can be recognized prenatally with ultrasound. Transplantation of mesenchymal stem cells (MSCs) has the potential to ameliorate skeletal damage. We report the clinical course of two patients with OI who received prenatal human fetal MSC (hfMSC) transplantation and postnatal boosting with same‐donor MSCs. We have previously reported on prenatal transplantation for OI type III. This patient was retransplanted with 2.8 × 106 same‐donor MSCs per kilogram at 8 years of age, resulting in low‐level engraftment in bone and improved linear growth, mobility, and fracture incidence. An infant with an identical mutation who did not receive MSC therapy succumbed at 5 months despite postnatal bisphosphonate therapy. A second fetus with OI type IV was also transplanted with 30 × 106 hfMSCs per kilogram at 31 weeks of gestation and did not suffer any new fractures for the remainder of the pregnancy or during infancy. The patient followed her normal growth velocity until 13 months of age, at which time longitudinal length plateaued. A postnatal infusion of 10 × 106 MSCs per kilogram from the same donor was performed at 19 months of age, resulting in resumption of her growth trajectory. Neither patient demonstrated alloreactivity toward the donor hfMSCs or manifested any evidence of toxicities after transplantation. Our findings suggest that prenatal transplantation of allogeneic hfMSCs in OI appears safe and is of likely clinical benefit and that retransplantation with same‐donor cells is feasible. However, the limited experience to date means that it is not possible to be conclusive and that further studies are required.

Human umbilical cord wharton's jelly stem cell (hWJSC) extracts inhibit cancer cell growth in vitro

Umbilical cord mesenchymal stem cells (MSCs) have been shown to inhibit breast cancer cell growth but it is not known whether this effect is specific to only breast cancer cells. We compared the effects of human Whartons jelly stem cell (hWJSC) extracts [conditioned medium (hWJSC‐CM) and cell lysate (hWJSC‐CL)] on breast adenocarcinoma (MDA‐MB‐231), ovarian carcinoma (TOV‐112D), and osteosarcoma (MG‐63) cells. The cells were treated with either hWJSC‐CM (50%) or hWJSC‐CL (15 µg/ml) for 48–72 h and changes in cell morphology, proliferation, cycle, gene expression, migration, and cell death studied. All three cancer cell lines showed cell shrinkage, blebbing, and vacuolations with hWJSC‐CL and hWJSC‐CM compared to controls. MTT and BrdU assays showed inhibition of cell growth by 2–6% and 30–60%, while Transwell migration assay showed inhibition by 20–26% and 31–46% for hWJSC‐CM and hWJSC‐CL, respectively, for all three cancer cell lines. Cell cycle assays showed increases in sub‐G1 and G2/M phases for all three cancer cell lines suggestive of apoptosis and metaphase arrest. AnnexinV‐FITC and TUNEL positive cells seen in TOV‐112D and MDA‐MB‐231 suggested that inhibition was via apoptosis while the presence of anti‐BECLIN1 and anti‐LC3B antibodies seen with MG‐63 indicated autophagy. Upregulation of pro‐apoptotic BAX and downregulation of anti‐apoptotic BCL2 and SURVIVIN genes were observed in all three cancer cell lines and additionally the autophagy genes (ATG5, ATG7, and BECLIN1) were upregulated in MG‐63 cells. hWJSCs possess tumor inhibitory properties that are not specific to breast cancer cells alone and these effects are mediated via agents in its extracts. J. Cell. Biochem. 113: 2027–2039, 2012.

Implanon® contraceptive implants: effects on carbohydrate metabolism

The objective of the study was to assess the possible differences in effects of Implanon and Norplant implants on carbohydrate metabolism. This is a 2-year open randomized study of 80 implant (Implanon and Norplant) acceptors. Oral glucose tolerance test (OGTT) was performed before implant insertion and at 6, 12, and 24 months after implant insertion. Glycosylated hemoglobin A(1)C was measured in fasting samples and plasma samples during OGTT were tested for glucose and insulin levels. There was a significant increase in the area under the curve for both glucose and insulin during OGTT within each group with increasing duration of use. However, there was no significant change in the fasting plasma glucose values. There was no significant difference in the carbohydrate parameters between the two groups during implant use, except for a minimal but statistically significant rise in fasting glycosylated hemoglobin A(1)C levels at 24 months in the Implanon group. Both implants appear to induce mild insulin resistance but no significant change in serum glucose levels. These alterations in carbohydrate metabolism should have no clinical significance in healthy women.

Levonorgestrel concentrations during 7 years of continuous use of Jadelle contraceptive implants.

Serum levonorgestrel concentrations were assayed in a multicenter, 7-year study of 199 users of Jadelle rod implants. We examined drug levels, patterns of changes, factors affecting drug levels, and concentrations at which pregnancies occurred. Mean levonorgestrel concentrations declined from 435 pg/mL at 1 month of use to 64% of that value (280 pg/mL) at the end of 3 years. Between the end of the third and fifth years neither mean nor median serum levels varied markedly. At 5 years the mean concentration was again 64% of the first months mean. Declining levels were observed thereafter through the end of 7 years when the mean, 224 pg/mL, was 52% of the 1-month value. Last measured drug concentrations of women who became pregnant during Jadelle use had mean and median values of 152 and 144 pg/mL, respectively, and a maximum value of 180 pg/mL. Analyses indicated ponderal index, body weight, duration of use, and a single clinical center were the most important variables affecting measured levonorgestrel levels. Approximately one-third of assays in the sixth and seventh years were found to be below 180 pg/mL, suggesting that Jadelle levonorgestrel implants would not maintain sufficiently high levels of effectiveness against pregnancy after 5 years and that heavier women would then be at greater risk of pregnancy.

Human umbilical cord wharton's jelly mesenchymal stem cells do not transform to tumor‐associated fibroblasts in the presence of breast and ovarian cancer cells unlike bone marrow mesenchymal stem cells

Human bone marrow mesenchymal stem cells (hBMMSCs) were shown to transform into tumor‐associated fibroblasts (TAFs) when in the vicinity of breast cancer tumors and played an important role in tumor enhancement and metastasis. In early human development MSCs migrating from the yolk sac and aorta‐gonad‐mesonephros (AGM) via the umbilical cord to the placenta and back to the fetal bone marrow were shown to get trapped in the gelatinous Whartons jelly of the umbilical cord. The common origin of the Whartons jelly MSCs and the finally homed hBMMSCs prompted us to evaluate whether hWJSCs are also involved in TAF transformation. hWJSCs and hBMMSCs were grown in the presence of breast and ovarian cancer cell conditioned medium (MDA‐TCM, TOV‐TCM) for 30 days. No changes were observed in the hWJSCs but the hBMMSCs transformed from short to thin long fibroblasts, their proliferation rates increased and CD marker expression decreased. The transformed hBMMSCs showed positive staining for the tumor‐associated markers FSP, VEGF, EGF, and Tn‐C. Real‐time PCR and multiplex luminex bead analysis showed upregulation of TAF‐related genes (FSP, FAP, Tn‐C, Tsp‐1, EGF, bFGF, IL‐6, α‐SMA, VEGF, and TGF‐β) for hBMMSCs with low expression for hWJSCs. The luciferase assay showed that hWJSCs previously exposed to MDA‐TCM or TOV‐TCM had no stimulatory growth effect on luciferase‐tagged MDA or TOV cells unlike hBMMSCs. The results confirmed that hWJSCs do not transform to the TAF phenotype and may therefore not be associated with enhanced growth of solid tumors making them a safe MSC for cell based therapies. J. Cell. Biochem. 113: 1886–1895, 2012.

Lipidomic analysis of human placental Syncytiotrophoblast microvesicles in adverse pregnancy outcomes

PROBLEM Syncytiotrophoblast microvesicles (STBM) are shed from placenta into the maternal circulation. STBM circulate in increased amounts in adverse pregnancies, e.g., preeclampsia and recurrent miscarriages (RM). Recently dysregulation of lipid metabolites has been proposed to be associated with their pathogenesis. Lipid composition of STBM in healthy and adverse pregnancies remains unknown. OBJECTIVE To determine lipid composition of STBM and whether STBM lipid composition differs in pathologic and normal pregnancies. STUDY DESIGN Patients with Preeclampsia (n = 6) or history of RM (n = 9) (>2 consecutive losses <20 weeks) and gestational age-matched normal pregnant controls (same number as cases) were recruited. STBM were prepared from placental explant culture supernatant. Lipid profiling of STBM was performed by mass spectrometry in combination with liquid chromatography. We quantified ∼200 lipids in STBM including (i) glycerophospholipids (phosphatidylcholine, PC; phosphatidylethanolamine, PE; phosphatidylinositol, PI; phosphatidylglycerol, PG; phosphatidylserine, PS; phosphatidic acid, PA); (ii) sphingolipids (sphingomyelin, SM; ceramide, Cer; Glucosylceramide, GluCer; ganglioside mannoside 3, GM3); (iii) free cholesterol and cholesteryl esters, CE. RESULTS The major lipid classes in STBM were SM, Chol, PS, PC and PI, along with PA and GM3 enrichments. SM/PC ratio showed a unique reversal (3:1) compared to that normally found in human cells or plasma. Level of total PS was significantly upregulated (p < 0.005) in preeclampsia patients, while PI (p < 0.0005), PA (p < 0.005), and GM3 (p < 0.05) were significantly downregulated. Similar trends were obtained in RM. CONCLUSIONS Differential lipid expression of STBM in preeclampsia or RM includes those that are implicated in immune response, coagulation, oxidative stress, and apoptosis.

Human umbilical cord Wharton's jelly stem cells and its conditioned medium support hematopoietic stem cell expansion ex vivo

Bone marrow mesenchymal stromal cells (BMMSCs) have been used as feeder support for the ex vivo expansion of hematopoietic stem cells (HSCs) but have the limitations of painful harvest, morbidity, and risk of infection to the patient. This prompted us to explore the use of human umbilical cord Whartons jelly MSCs (hWJSCs) and its conditioned medium (hWJSC‐CM) for ex vivo expansion of HSCs in allogeneic and autologous settings because hWJSCs can be harvested in abundance painlessly, are proliferative, hypoimmunogenic, and secrete a variety of unique proteins. In the presence of hWJSCs and hWJSC‐CM, HSCs put out pseudopodia‐like outgrowths and became highly motile. Time lapse imaging showed that the outgrowths helped them to migrate towards and attach to the upper surfaces of hWJSCs and undergo proliferation. After 9 days of culture in the presence of hWJSCs and hWJSC‐CM, MTT, and Trypan blue assays showed significant increases in HSC numbers, and FACS analysis generated significantly greater numbers of CD34+ cells compared to controls. hWJSC‐CM produced the highest number of colonies (CFU assay) and all six classifications of colony morphology typical of hematopoiesis were observed. Proteomic analysis of hWJSC‐CM showed significantly greater levels of interleukins (IL‐1a, IL‐6, IL‐7, and IL‐8), SCF, HGF, and ICAM‐1 compared to controls suggesting that they may be involved in the HSC multiplication. We propose that cord blood banks freeze autologous hWJSCs and umbilical cord blood (UCB) from the same umbilical cord at the same time for the patient for future ex vivo HSC expansion and cell‐based therapies. J. Cell. Biochem. 113: 658–668, 2012.