Freezing Amnion-Derived Stem Cells in Xeno-Free Media Feasible


Amnion-derived stem cells show a dizzying set of regenerative properties, at least in the laboratory (see Miki T, Stem Cell Res Ther 2011, 2:25; Miki T, Grubbs B, J Obstet Gynaecol Res 2014, 40:360-8). Bringing these cells into the clinic will definitely take a lot more work, but a new paper examines a culture system that does not use any animal products. Such culture systems (known as “xeno-free) are vitally important if stem cells are going to be used in human trials.

When stem cells are grown in culture, typically serum from animal blood is used to provide the cells with the growth factors and things they need to kick the cells into the growth phase. However, occasionally, some animal-based products have animal viruses that can infect human cells with unpredictable consequences (see Karlsson JOM, Toner M, Biomaterials 1996, 17:243-56). Also, cells grown in animal-based culture media can have animal proteins around them that are very hard to get rid of. Implanting such cells into a human patient would cause their immune system to react against the animal proteins. Such immune responses might result in something as innocuous as itching at the site of injection or as dangerous as anaphylactic shock. Therefore, growing stem cells under xeno-free conditions is important for many stem cell applications.  Expanding and preserving stem cells under so-called Good Manufacturing Pracctices (GMP) is essential for their use in human patients.

However, now we have just opened another can of worms. If all the characterizations of stem cells have been under culture conditions that utilize animal-based culture media, will the cells have the same capabilities if grown under xeno-free conditions?

Herein is the reason why this paper by Toshio Miki from the Keck School of Medicine at the University of Southern California and his colleagues is so important. In this paper, which was published in the journal Stem Cell Research and Therapy (Stem Cell Research & Therapy 2016, 7:8 doi:10.1186/s13287-015-0258-z), Miki and others examined the characteristics of amniotic stem cells grown in xeno-free media (see Mitry RR, Lehec SC, Hughes RD, Methods Mol Biol 2010, 640:107-13; Polchow B, et al. J Transl Med 2012, 10:98; Stacy GN, Masters JR, Nat Protoc 2008, 3:1981-89; Miki T, et al. Curr Protoc Stem Cell Biol 2010, Chapter 1:Unit 1E.3) and compared them to stem cells grown in standard culture media. This is a very important exercise, since amniotic stem cells are easily accessible as source of material for regenerative treatments that can be banked and immunotype matched for clinical applications.

Miki and others isolated human amniotic epithelial cells from newborn babies with the consent of their parents and then stored the cells at −160 °C in one of five commercially available culture media. Miki and his group used cells frozen in standard media containing fetal bovine serum as controls to which all the other cells were compared. Then they thawed the cells, and tested their viability, mitochondrial integrity, and senescence status (tendency to fall asleep in culture and stop growing). They also examined gene expression profiles in these cells by using quantitative real-time PCR. Flow cytometry was used to identify the stem cell surface markers.

The results were encouraging and interesting. There were no significant differences in viability and growth in cells grown and preserved in xeno-free media versus standard cryopreservation medium. Additionally, comparisons of the cells grown in the different cryopreservation media did not reveal significant differences in the senescence status, mitochondria, or overall morphology of the cells. The upshot is that the cells preserved in standard or xeno-free media looked and grew the same after being thawed. There were some differences in the expression of stem cell marker genes (e.g., OCT4, SOX2, and NANOG) and a particular cell surface marker (TRA1-60) following cryopreservation in different xeno-free media. However, it turns out these differences were slight and, overall, not statistically significant. Again, the upshot is that there were small differences in gene expression, but these differences did not amount to a hill of beans.

Miki and his colleagues have nicely shown that cryopreserving amnion-derived stem cells in xeno-free media is feasible and does not affect the characteristics of the cells. This paper also suggests that such xeno-free media can be used to establish a bio-bank of human amnion-derived stem cells for future clinical application.

Well that’s one hurdle vaulted. Now Miki and others need to figure out which of the xeno-free media is the best and optimize that medium to for improved preservation of stem cell-like characteristics in these cells.

Stem Cells Derived From Amniotic Tissues Have Immunosuppressive Properties


Ever since they were first isolated, amnion-based stem cells have been considered promising candidates for cell therapies because of their ease of access, plasticity, and absence of ethical issues in their derivation and use. However, a Japanese research team has discovered that stem cells derived from human female amnion also have the ability to suppress the inappropriate activation of the immune system and that there are straight-forward ways to enhance their immunosuppressive potential.

The amniotic membrane is a three-layered structure that surrounds the baby and suspends it in amniotic fluid. Amniotic fluid acts as a protective shock-absorber, a lubricant and an important physiological player in the life of the embryo and fetus. Because the fetus is a privileged entity that escapes attack from the mother’s immune system, researchers have been very interested in determining the immunological properties of the amnion cells.

“The human amniotic membrane contains both epithelial cells and mesenchymal cells,” said study co-author Dr. Toshio Nikaido, Department of Regenerative Medicine, Graduate School of Medicine and Pharmaceutical Sciences at the University of Toyama. “Both kinds of cells have proliferation and differentiation characteristics, making the amniotic membrane a promising and attractive source for amnion-derived cells for transplantation in regenerative medicine. It is clear that these cells have promise, although the mechanism of their immune modulation remains to be elucidated.”

In this study by Nikaido and his coworkers, amnion-derived cells inhibited natural killer cell activity and induced white blood cell activation. Nikaido reported that he and his colleagues saw the amnion-derived cells increase production of a molecule called interleukin-10 (IL-10).

“We consider that IL-10 was involved in the function of amnion-derived cells toward NK cells,” explained Dr. Nikaido. “The immunomodulation of amnion-derived cells is a complicated procedure involving many factors, among which IL-10 and prostaglandin E2 (PGE2) play important roles.”

Molecules called “prostaglandins,” such as PGE2, mediate inflammation, smooth muscle activity, blood flow, and many other physiological process. In particular, PGE2 exerts important effects during labor and stimulates osteoblasts (bone-making cells) to release factors that stimulate bone resorption by osteoclasts. PGE2 also suppresses T cell receptor signaling and may play a role in the resolution of inflammation.

When Nikaido and others used antibodies against PGE2 and IL-10, they removed the immunosuppressive effects of the amnion-derived cells on natural killer cells. These data imply that these two factors contribute to the immunosuppressive abilities of amnion-derived cells.

“Soluble factors IL-10 and PGE2 produced by amnion-derived cells may suppress allogenic, or ‘other’ related immune responses,” concluded Dr. Nikaido. “Our findings support the hypothesis that these cells have potential therapeutic use. However, further study is needed to identify the detailed mechanisms responsible for their immodulatory effects. Amnion-derived cells must be transplanted into mouse models for further in vivo analysis of their immunosuppressive activity or anti-inflammatory effects.”

Given the levels of autoimmune diseases on the developed world, these results could be good news for patients who suffer from diseases like Crohn’s disease, systemic lupus erythematosus, or rheumatoid arthritis. While more work is needed, amnion-based cells certainly show promise as immunosuppressive agents.

The study will be published in a future issue of Cell Transplantation.

Bone-Making Ability of Amniotic Epithelial Cells


Human amniotic epithelial cells (hAECs) develop from the embryo as early as eight days after fertilization. hAECs have been shown to possess remarkable plasticity, or the ability to form many cell types. Mattioli and others showed that AECs from sheep have capacity to differentiate into bone cells (see Cell Biol Int 2012;36:7-19). Therefore, it is possible that these cells could be developed into a source of cells for regenerative treatments.

Steve Shen from the Shanghai Jiao Tong University School of Medicine and his coworkers have examined the ability of cultured hAECs to make bone in culture, and then applied their techniques to an animal system to test the ability of cultured hAECs to restore tooth sockets and facial bone.

To begin, hAECs were isolated from healthy mothers who had undergone childbirth by cesarean section. These cells were cultured in a standard cell culture medium (DMEM/F12 for those who are interested), expanded in culture, and then subjected to flow cytometry to isolate those cells with all the right cell surface markers.

These cells wee then placed into a specialized culture medium designed to convert mesenchymal stem cells into bone-making cells. This osteogenic induction medium was changed every three days for 21 days.  The figure below shows how well the bone-induction worked.  A red stain called alizarin red binds to calcium and therefore stains forming bone matrices rather well.  As you can see in the panel labeled “G” below, the hAECs grown in the osteogenic medium stain red rather well, which shows that they are making bone.

Characterization of hAECs in vitro. (A, B): hAECs at passages 0 and 1 displayed a cobblestone-like morphology. (C): Some hAECs changed into fibroblast-like cells after 7 days of osteoblastic culture. (D): hAECs showed significant morphological changes and settled on superimposed layers after 21 days of osteoblastic culture. (E): Cell proliferation of hAECs at passage 1 was significantly higher than at passages 0 and 5 from day 4 to day 12; hAECs at passage 5 displayed the lowest proliferation rate from day 6 to day 14 (☆, p < .05). (F): Flow cytometry analysis showed that hAECs expressed CD44, CD90, CD105, and SSEA-4 and did not express CD34, CD45, and HLA-DR. Values represent the percentages of all assessed cells positively stained by the indicated antigens (bottom of each graph). Nonspecific fluorescence was determined as the blank control using isotype-matched monoclonal antibodies (PE blank, FITC blank). (G): Representative images of microscopic and general photographs for ALP and ARS staining in osteogenic and control groups indicated the osteogenic differentiation of hAECs in vitro. Scale bar = 200 μm. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S; FITC, fluorescein isothiocyanate; hAEC, human amniotic epithelial cell; OD, optical density; p, passage; PE, phycoerythrin.
Characterization of hAECs in vitro. (A, B): hAECs at passages 0 and 1 displayed a cobblestone-like morphology. (C): Some hAECs changed into fibroblast-like cells after 7 days of osteoblastic culture. (D): hAECs showed significant morphological changes and settled on superimposed layers after 21 days of osteoblastic culture. (E): Cell proliferation of hAECs at passage 1 was significantly higher than at passages 0 and 5 from day 4 to day 12; hAECs at passage 5 displayed the lowest proliferation rate from day 6 to day 14 (☆, p < .05). (F): Flow cytometry analysis showed that hAECs expressed CD44, CD90, CD105, and SSEA-4 and did not express CD34, CD45, and HLA-DR. Values represent the percentages of all assessed cells positively stained by the indicated antigens (bottom of each graph). Nonspecific fluorescence was determined as the blank control using isotype-matched monoclonal antibodies (PE blank, FITC blank). (G): Representative images of microscopic and general photographs for ALP and ARS staining in osteogenic and control groups indicated the osteogenic differentiation of hAECs in vitro. Scale bar = 200 μm. Abbreviations: ALP, alkaline phosphatase; ARS, alizarin red S; FITC, fluorescein isothiocyanate; hAEC, human amniotic epithelial cell; OD, optical density; p, passage; PE, phycoerythrin.

When hAECs were subjected to gene expression assays, it was clear that the cells grown in the osteogenic medium expressed a whole host of bone-specific genes.  Therefore, the formation of bone was not a fluke, since these cells not only concentrated calcium, but also increased their expression of bone-specific genes (these include osterix, Runx2, Alkaline phosphatase, Collagen I, and osteoprotegerin).

Additionally, those hAECs grown in osteogenic medium stopped forming sheets of cells and began to grow as individual cells.  This is an important transformation for the synthesis of bone because bone-making cells secrete a bone-specific protein matrix upon which calcium phosphate-based crystals deposit to form bone.  If the cells were organized in sheets, then bone deposition would not be possible.  When isolated from amniotic membranes, hAECs grow as sheets of cells that are known as epithelia.  In order to become bone-making cells, the hAECs must undergo an “epithelial-to-mesenchymal transformation” or EMT.  This requires turning on new genes and turning off others.  Gene expression assays of hAECs grown in the osteogenic medium, show extensive evidence of EMT.  For example, a protein called E-cadherin is essential for cells growing in sheets, because it helps cells stick to each other.  However, in hAECs grown in the osteogenic medium, E-cadherin expression was quite low.  Also, a protein called vimentin is highly expressed during EMT, and the hAECs grown in osteogenic medium showed high expression of vimentin.  Thus, these hAECs were undergoing all the necessary changes in order to become bone-making cells,, making all the right genes, and made bone in culture to boot.

This is certainly interesting, but can these hAECs repair bone in a living animal?  Shen’s group tried that very experiment.  The hAECs that were grown in the osteogenic medium were loaded on tricalcium phosphate scaffolds and implanted into rodents with tooth socket lesions.  Control animals were implanted with tricalcium phosphate scaffolds without cells.  The scaffold with cells significantly increased bone formation in the rodents, and showed much more infilling with mineralized tissue.  There was also extensive evidence of the formation of new vasculature and wandering cells called macrophages that are important for the degradation of the implanted scaffold required for new bone formation.  Tissue samples were examined 4 to 8 weeks after the implants were placed.

In vivo healing process in alveolar defect at 4 and 8 weeks postoperatively. (A): Representative three-dimensional micro-computed tomography (CT) reconstruction images of hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 1 mm. (B): Micro-CT parameters acquired among β-TCP scaffold in vitro, hAECs+β-TCP scaffold in vivo, and β-TCP scaffold alone in vivo at 4 and 8 weeks postoperatively (☆, p < .05). (C): Hematoxylin and eosin staining of the rat alveolar defect at 4 and 8 weeks postoperatively revealed more active new bone formation in the EXP group than in the CTR group (×50 and ×200 magnification). Alveolar defect treated with hAECs+β-TCP scaffold exhibited a more mature lamellae-bone formation at 8 weeks postoperatively. Scale bar = 200 μm. (D): ANA-positive cells, visible as green fluorescence in the nuclei, were observed within the newly deposited OCN, and OPN-positive bone tissue, visible as red fluorescence, in the EXP group at 4 weeks postoperatively, indicating a mature osteoblastic function of these hAEC-derived cells. Scale bar = 50 μm. (E): Representative images of immunohistochemical staining of sections with anti-VEGF antibody and anti-CD68 antibody in hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 200 μm. (F): The histomorphometric quantification of the relative new bone area, VEGF-positive area, and CD68-positive area showed that more bone tissue regeneration was observed in the EXP group than in the CTR group at 4 and 8 weeks postoperatively. The positive signal of VEGF and CD68 in the EXP group was much weaker at 4 weeks postoperatively and became more intense at 8 weeks postoperatively compared with the CTR group (☆, p < .05). Abbreviations: ANA, anti-nuclear antibody; BV/TV, bone volume/tissue volume ratio; BMD, bone mineralization density; hAECs, human amniotic epithelial cells; OPN, osteopontin; post op, postoperatively; SMI, structure model index; Tb.Th., trabecular thickness; Tb.N., trabecular number; Tb.Sp., trabecular separation; β-TCP, β-tricalcium phosphate; VEGF, vascular endothelial growth factor.
In vivo healing process in alveolar defect at 4 and 8 weeks postoperatively. (A): Representative three-dimensional micro-computed tomography (CT) reconstruction images of hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 1 mm. (B): Micro-CT parameters acquired among β-TCP scaffold in vitro, hAECs+β-TCP scaffold in vivo, and β-TCP scaffold alone in vivo at 4 and 8 weeks postoperatively (☆, p < .05). (C): Hematoxylin and eosin staining of the rat alveolar defect at 4 and 8 weeks postoperatively revealed more active new bone formation in the EXP group than in the CTR group (×50 and ×200 magnification). Alveolar defect treated with hAECs+β-TCP scaffold exhibited a more mature lamellae-bone formation at 8 weeks postoperatively. Scale bar = 200 μm. (D): ANA-positive cells, visible as green fluorescence in the nuclei, were observed within the newly deposited OCN, and OPN-positive bone tissue, visible as red fluorescence, in the EXP group at 4 weeks postoperatively, indicating a mature osteoblastic function of these hAEC-derived cells. Scale bar = 50 μm. (E): Representative images of immunohistochemical staining of sections with anti-VEGF antibody and anti-CD68 antibody in hAECs+β-TCP scaffold (EXP) and β-TCP scaffold (CTR) at 4 and 8 weeks postoperatively. Scale bar = 200 μm. (F): The histomorphometric quantification of the relative new bone area, VEGF-positive area, and CD68-positive area showed that more bone tissue regeneration was observed in the EXP group than in the CTR group at 4 and 8 weeks postoperatively. The positive signal of VEGF and CD68 in the EXP group was much weaker at 4 weeks postoperatively and became more intense at 8 weeks postoperatively compared with the CTR group (☆, p < .05). Abbreviations: ANA, anti-nuclear antibody; BV/TV, bone volume/tissue volume ratio; BMD, bone mineralization density; hAECs, human amniotic epithelial cells; OPN, osteopontin; post op, postoperatively; SMI, structure model index; Tb.Th., trabecular thickness; Tb.N., trabecular number; Tb.Sp., trabecular separation; β-TCP, β-tricalcium phosphate; VEGF, vascular endothelial growth factor.

hAECs are typically not recognized by the immune system as foreign and they also have anti-scarring capabilities.  Not only can they effectively form bone in culture, but they can also repair an alveolar defect in a rodent model.  Clearly these cells show promise for clinical applications.  However, before they can be used in the clinic more has to be known about their culture conditions, how many cells are required for transplantation, the best cell dose, at what passages they need to be used, and so on.  Thus, this paper represents a good start for hAECs, but they have to be better understood before they can come to the clinic.

Human Amniotic Epithelial Cells Modulate Tooth Socket Restoration in Rats


Human amniotic epithelial cells have the capacity to differentiate into several different cell types. To that list, we can now add bone.

A study from Steve G.F. Shen at his colleagues at the Shanghai Jiao Tong University School of Medicine, Shanghai, China has used human amniotic epithelial cells to regenerate the tooth sockets in laboratory animals.

The first set of experiments examined the ability of human amniotic epithelial cells (hAECs) to form bone under controlled laboratory conditions. Then hAECs were loaded into artificial scaffolds that were then placed into the mouths of rats with tooth socket defects.

In culture, hAECs expressed bone-specific genes 10-14 days after induction. The cells also changed shape and made bone-specific proteins. When implanted into rat tooth sockets, the hAECs were embedded in a scaffold imbued with growth factors known to induce bone differentiation. These implants improved bone regeneration by directly participating in bone repair of the tooth socket defect. They also had an additional benefit in that they modulated the localized immune response against the implanted scaffolds. This immune response modulation augmented regeneration of the tooth sockets and allowed the implanted cells to get on with the job of fixing the surrounding bone without dealing with insults from the immune system.

This study has provided the first evidence that hAECs exhibit direct involvement in new bone regeneration and a localized modulatory influence on the early tissue remodeling process. These cells indirectly contributed to the bone-making process in the alveolar defect. Altogether, these results imply the potential clinical use of hAECs as an alternative stem cell-based for restoring tooth socket deformities.

Radio Interview About my New Book


I was interviewed by the campus radio station (89.3 The Message) about my recently published book, The Stem Cell Epistles,

Stem Cell Epistles

It has been archived here. Enjoy.

Human Amniotic Epithelial Cells – Remarkable Possibilities for a Small Price


My apologies to my readers for my inactivity. Many deadlines make for less blogging. Nevertheless, I hope to get back to a more regular blogging schedule once things quiet down a bit.

Today’s entry is about a fascinating group of cells found in the extraembryonic membranes of the fetus known as the amnion. The amniotic sac is a thin, transparent pair of membranes that is actually rather tough. This sac holds the fetus until shortly before birth. In inner membrane of the amnion sac contains the amniotic fluid and fetus and the outer membrane, the chorion, surrounds the amnion and is part of the placenta.

The amniotic membrane contains a remarkable cell type known as amniotic epithelial cells or hAECs (the “h” is for human). Upon isolation after birth, the amnion membrane and manually separated from the chorion membrane and washed in a saline (salt) solution in order to remove all the blood. Then the epithelial cells are liberated from the basement membrane upon which they sit by a product called TrypZean. TrypZean is a recombinant trypsin, which is very clean and devoid of animal products. Trypsin is one of the enzymes in your digestive system that degrades proteins. By expressing the human trypsin gene in bacteria and purifying the protein, Sigma-Aldrich corporation can sell it for a profit to scientists for various procedures.

A single amnion membrane can yield in the vicinity of 120 million viable hAECs, which can be maintained in serum-free culture conditions. After being grown for some time, hAECs will have normal chromosome compositions and will also maintain chromosomes that have nice, long ends (telomeres). This indicates that the cells are healthy and dying while they grow in culture (see Murphy et al., Current Protocols in Stem Cell Biology, 2010; Chapter 1: Unit 1E.6). .

In culture,. hAECs do not grow like weeds. Mesenchymal stem cells (MSCs) tend to grow better than their hAEC brethren, but hAECs possess a remarkable ability to differentiate into a wide variety of different cell types. Sivakami Ilancheran in the laboratory of Martin Pera at the University of Monash in Clayton, Australia showed that hAECs were able to differentiate into heart muscle, skeletal muscle, bone, fat cells, pancreatic cells, liver, and at least two kinds of nerve cells. Also, when injected into mice, hAECs never formed tumors (Ilancheran et al., Biology of Reproduction 77 (2007): 577-88). Murphy and others have also shown that hAECs can be isolated after collection and stored for clinical therapies.

Given that hAECs are accessible, what are they good for? When it comes to regenerative medicine, preclinical studies with hAECs have produced very solid results that may pave the way for other studies.

HAECs can differentiate into lung cells and this feature makes them an attractive candidate for lung diseases. Lung diseases cause inflammation of the lung and scarring that decreases overall lung capacity. Cystic fibrosis, acute respiratory distress syndrome, chronic obstructive pulmonary disease, pulmonary fibrosis, pulmonary edema, and pulmonary hypertension are all lung diseases that could potentially be treated with hAECs.

In animal models of lung disease, particular chemicals are given to the animal that damage the lung. The wounded lung tissue initiates inflammation that brings white blood cells into the lung that augment the lung damage, which results in lung scarring. If hAECs are given to mice whose lungs have been damaged by the anti-cancer drug bleomycin, the signs of inflammation and the genes normally expressed during inflammation fade away. There is also less scarring in the lungs and the functional recovery of these animals is significantly better than those animals that do not receive hAECs (Murphy et al., Cell Transplantation 2011 20(6): 909-23). In fact, hAECs can differentiate into lung cells and integrate into lung tissue. The significance of this is not lost on respiratory specialists who treat patients with cystic fibrosis. Cystic fibrosis patients lack a functional copy of a ion transport protein and poor ion transport cause the production of thick, sticky mucous that clogs up the lung pathways and causes patients to suffocate to death. However, hAECs can differentiate into lung cells that express this ion transporter. Therefore, hAECs could be a potential treatment for cystic fibrosis. Clearly hAECs have great potential for tissue engineering applications with lung disease.

Lungs are not the only organ that hAECs can help heal. These cells can also differentiate into pancreatic insulin-making cells. In the laboratory, Wei and coworkers succeeded in stimulating hAECs to secrete insulin and express the main sugar transport protein found in pancreatic insulin-secreting cells (Wei et al., Cell Transplantation 2003 12(5): 545-552). When transplanted into diabetic mice, hAECs normalize their blood sugar levels and their weights returned to normal. This shows that hAECs might represent a major breakthrough in the management of diabetes.

Clearly these cells, which come from a tissue that is normally thrown out after birth, are brimming with possibilities for regenerative medicine. Hopefully more research will produce even more possibilities.