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. 2022 Aug 23;46(12):1999–2008. doi: 10.1002/cbin.11892

Strategies for immortalisation of amnion‐derived mesenchymal and epithelial cells

Aneesa Ansari 1,2,✉, Kate M Denton 1,2, Rebecca Lim 3,4
PMCID: PMC9804755  PMID: 35998259

Abstract

Mesenchymal (human amniotic mesenchymal stem cell [HAMSC]) and epithelial cells (human amnion epithelial cell [HAEC]) derived from human amniotic membranes possess characteristics of pluripotency. However, the pluripotency of HAMSC and HAEC are sustained only for a finite period. This in vitro cell growth can be extended by cell immortalisation. Many well‐defined immortalisation systems have been used for artificially overexpressing genes such as human telomerase reverse transcriptase in HAMSC and HAEC leading to controlled and prolonged cell proliferation. In recent years, much progress has been made in our understanding of the cellular machinery that regulates the cell cycle when immortalised. This review summarises the current understanding of molecular mechanisms that contribute to cell immortalisation, the strategies that have been employed to immortalise amnion‐derived cell types, and their likely applications in regenerative medicine.

Keywords: amnion epithelial cells, hTERT, immortalisation, mesenchymal cells, placenta

Abbreviations

AM

amniotic membrane

Bmi‐1

a polycomb group of genes

CDK

cyclin‐dependent kinases

CDKi

CDK inhibitors

DDR

DNA damage response

HAEC

human amnion epithelial cells

HAMSC

human amniotic mesenchymal stem cells

HPV‐16 E6/E7

human papillomavirus type 16 E6 and E7

hTERT

human telomerase reverse transcriptase

Rb

retinoblastoma protein

1. INTRODUCTION

Stem‐like cells derived from human amniotic membranes, such as the epithelial (human amnion epithelial cell [HAEC]) and mesenchymal cells (human amniotic mesenchymal stem cell [HAMSC]) are already in clinical trials. To date, 20 clinical trials have been registered for HAECs and two for HAMSC on ClinicalTrials.gov. Consequently, there is a considerable and increasing demand for these cells. Although, they can be easily obtained from discarded placental tissues and the cost of extraction is low, there is the issue of low in vitro proliferation capacity and high variability of the cells (Antoniadou & David, 2016; Murphy et al., 2010). There are no known cell lines that have the amniotic‐derived cell‐like characteristics that have unlimited growth and high reproducibility capacity. By using standard strategies, HAEC and HAMSC are typically immortalised from healthy primary cells isolated from the amniotic membrane of the discarded placenta from caesarian patients (Teng  et al., 2013; Zhou et al., 2013). The purpose of this paper is to discuss the existing techniques used for the immortalisation of HAMSC and HAEC and provide an overview of their application in clinical application and in vitro research.

2. HUMAN AMNION‐DERIVED CELLS

The amniotic membrane is the innermost layer of the fetal membranes found attached to the placenta (Figure 1a). The unique properties of the amniotic membrane, such as immune‐modulation (Garfias et al., 2011), anti‐inflammatory activity (Marsh et al., 2017), low immunogenicity (Kubo et al., 2001) and antibacterial capacity (Tehrani et al., 2013), are beneficial to clinical applications such as skin transplantation (Liang et al., 2020), reconstruction of conjunctival and corneal surfaces (Gomes et al., 2003) and treatments of traumatic wounds and open ulcers (ElHeneidy et al., 2016). The amniotic membrane consists of two major cell types, HAEC, which forms a continuous monolayer that is in direct contact with the amniotic fluid, and the HAMSC, which is sparingly distributed just below the amnion epithelial cells within the underlying avascular collagenous stroma (Insausti et al., 2010; Parry & Strauss, 1998) (Figures 1 and 2). Both the HAEC and HAMSC express the cell surface markers associated with stem cells. These include the Tumour Rejection Antigens 1–60 and 1–81, molecular markers of pluripotency like Oct3/4, Sox2 and Stage‐Specific Embryonic Antigens 3 and 4 (Alviano et al., 2007; Insausti et al., 2010; Miki et al., 2005; Parolini et al., 2008). HAEC and HAMSC have the ability not to induce tumorigenesis upon transplantation (Li et al., 2019; Yang et al., 2018). Moreover, they suppress inflammation, and reportedly reduce the proliferation of the T‐ and B‐cells in vitro (Li et al., 2019; Vosdoganes et al., 2011). HAEC do not express human leukocyte antigen class II antigens and avoids immunological rejection (Banas et al., 2008; Li et al., 2005). These unique characteristics make amniotic membrane a suitable source for cell‐based therapies. However, amnion‐derived cells are known for their poor expansion potential and approaches senescence easily (Murphy et al., 2010; Portmann‐Lanz et al., 2006; Teng et al., 2013). Senescent cells typically appear distended, with enlarged or multiple nuclei, prominent golgi apparatus and vacuolated cytoplasm (Sharpless & Sherr, 2015) (Figure 2). In senescent cells, the activity of lysosomal mass and/or autophagy is also increased, and epigenetic changes like senescence‐associated heterochromatin foci are observed, with alterations in secretome composition. This is also referred to as a senescence‐associated secretory phenotype (Hernandez‐Segura et al., 2018; Xue et al., 2007). In comparison to HAMSC, HAECs are more challenging to maintain in culture because of their heterogeneity in relation to plastic adherence (Miki et al., 2005; Parolini et al., 2008). Regardless, cellular senescence remains a challenge for both HAEC and HAMSC derived from the amniotic membrane because of accumulated DNA damage or shortened telomeres. This is a challenge that must be overcome if these cell types are to be used clinically.

Figure 1.

Figure 1

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Placenta and amniotic membrane. (a, b) Amnion membrane being peeled from the placenta and placed on a petri dish. (b) The amniotic membrane consists of five layers, namely, epithelial (containing the epithelial cells [HAEC]), basement membrane, compact layer, fibroblastic layer (containing the mesenchymal cells [HAMSC]) and spongy layer. HAEC, human amnion epithelial cell; HAMSC, human amniotic mesenchymal stem cell.

Figure 2.

Figure 2

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Human amnion mesenchymal (HAMSC) and epithelial cells (HAEC). (a, c) Shows HAMSC and HAECs, respectively, before the first passage while (b) and (d) shows HAMSC and HAEC, respectively after passage 6.

3. CELL CYCLE REGULATION, CONTROL AND SENESCENCE

To date, 30 cyclins and >20 cyclin‐dependent kinases (CDK) have been identified in mammalian cells, but only a few are involved in cell cycle regulation (Malumbres & Barbacid, 2005; Malumbres et al., 2009). These cyclin/CDK complexes perform special functions in the cell cycle and influence assembly and activation events. The cell cycle is usually associated with the activation of the p53/p21 pathways and suppression of the cell division cycle 25 phosphatase activity, which governs cell cycle arrest (Vermeulen et al., 2003). In addition to cyclin binding, CDK activity is also regulated by the phosphorylation/dephosphorylation equilibrium of the cell cycle inhibitory proteins called the CDK inhibitors (CDKi) (Malumbres & Barbacid, 2005; Mohan & Asakura, 2017). Based on the identity and structure of the CDK target, the CDKi is divided into the Ink4 family and Cip/Kip family. The Ink4 family comprises p15, p16, p18 and p19/p14 that specifically inhibit the CDK4/6 whereas the Cip/Kip family proteins include p21, p27 and p57 which inhibit CDK1/2 (Mohan & Asakura, 2017; Vermeulen et al., 2003) (Figure 3).

Figure 3.

Figure 3

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Schematic diagram of the cell cycle. The cell cycle is divided into G1, S, G2 and M phase. The cyclins and the CDK help in promoting the cell cycle progression. DNA damage, cell injury and hypoxia trigger p53 which activate p21. P21, in turn, inactivates cyclin D and E. P16, p15, p18 and p19 also inhibit cyclin D and E. Moreover, p21, p27 and p57 inhibit cyclin B and A. The inhibition of the cyclin also inhibits the different phases of the cell cycle. CDK, cyclin‐dependent kinases; G0, resting phase; G1, gap 1; G2, gap 2; M, mitotic phase; S, synthesis.

The progression of the cell through the G1 (gap 1 or preparatory phase), S (synthesis or phase where DNA replicates), G2 (gap 2 or cell prepares to divide) and M (mitotic phase) phases can also be hindered by DNA damage, cell injury and even contact inhibition of growing cells (Puliafito et al., 2012) (Figure 3). The common mechanisms of cell arrest are the DNA damage response (DDR) and the p53/p21 pathways. The DDR pathway activates transcription tumour suppressor p53 which upregulates p21 at senescence. This p21, in turn, arrests cell cycle progression at the G1/S and G2/M transitions by inhibiting the CDK4/6 (cyclin D) and CDK2 (cyclin E) complexes (Bekker‐Jensen & Mailand, 2010). The DDR pathway is activated by DNA damage in the form of shortened and/or deprotected telomeres, DNA double‐strand breaks or collapsed replication forks (Bekker‐Jensen & Mailand, 2010). Once the DDR pathway is activated, the cell undergoes senescence and remains unrepaired (Khosla et al., 2020) (Figure 3).

4. MECHANISM FOR ESCAPING CELL SENESCENCE

Reference senescence refers to a stable cell cycle arrest, which relies on the permanent activation of the DDR pathway (Pack et al., 2019; Rossiello et al., 2014). If a cell bypasses or overcomes this cell cycle arrest, the cell then becomes ‘immortalised’ (Abbadie et al., 2017). P53 is the main inducer of reference senescence (Mijit et al., 2020). P53 plays a role as a transcription factor and maintains the genetic stability. Following DNA damage, p53 is activated and tries to hinder proliferation by inducing gene expression that leads to growth arrest such as p21 and p16 (Mijit et al., 2020). P21 and p16 are CDKi that bind specifically with CDK4/6 (cyclin D) complexes and controls suppression of retinoblastoma protein (Rb). When these complexes are functional, Rb is phosphorylated and cannot interact with the E2F family of transcription factors and this, in turn, activates the target genes for promoting the G1/S transition. When the cyclin/CDK complexes are inhibited by p16, Rb is hypophosphorylated; it interacts with E2F and arrests the cell cycle at the G1/S transition (Giacinti & Giordano, 2006; Mijit et al., 2020) (Figure 4). Therefore, inactivating p53 and Rb individually or combinatorially can lead to cellular immortalisation (Rovinski & Benchimol, 1988; Sage et al., 2000; Shay et al., 1991).

Figure 4.

Figure 4

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Difference between the cell‐cycle progression of normal and immortalised cell. In normal epithelial cells, pRb controls the expression of different proteins needed for the cell‐cycle. This pRb binds with the E2F transcription factor protein. The cyclin D/CDK4/6 is activated in the presence of growth factors, which leads to Rb phosphorylation and releases the E2F. E2F drives the expression protein for the S‐phase progression. P16, p18 and p19 regulate the levels of cyclin D/CDK in the cell providing a feedback to the cell whether or not to proliferate any further. P27 regulates the activation of cyclin E. p14/INK4a regulates the activity of ubiquitin ligase which in turn regulates the activity of the p53 at a level below that required for the cellular arrest or apoptosis. The immortalisation was achieved in two ways: HPV‐16 E6/E7 and hTERT gene insertion. In the case of HPV‐16 E6/E7, the E7 protein binds and degrades pRb and helps the E2F to stimulate the S‐phase cell cycle. Moreover, p14 inactivates Mdm2 and increases the p53 level. This is corrected by the E6 protein which binds with p53 and degrades it, therefore preventing growth arrest and/or apoptosis. In the case of hTERT, once the inserted gene is stabilised, the telomere erosion is prevented and helps in continuous cell growth. CDK, cyclin‐dependent kinases; E2F, genes for transcription factors; hTERT, human telomerase reverse transcriptase; HPV E6/E7, human papillomavirus E6 and E7 oncogenes; pRb, phosphorylation of retinoblastoma protein; S, synthesis.

Another common approach is the introduction of human telomerase reverse transcriptase (hTERT). HTERT is expressed in only a few cell types, including germ lines (Rudolph et al., 1999; J. Shay & Bacchetti, 1997), and the introduction of this gene restores telomerase activity and prevents telomere erosion (Bodnar et al., 1998; Nakayama et al., 1998; Vaziri & Benchimol, 1998). This was also observed when primary human keratinocytes were transfected with human papillomavirus type 16 E6 proteins, where p53 was degraded and telomerase activity was restored (Howie et al., 2009; Stöppler et al., 1997). The cellular proto‐oncogene c‐myc is also found to induce telomerase activity and immortalise primary prostate epithelial cells (Gil et al., 2005). C‐myc acts as a promoter for hTERT. The hTERT promoter contains various c‐myc‐binding sites (CACGTG) through which c‐myc can activate telomerase (Greenberg et al., 1999; Wu et al., 1999) and promote cell proliferation in the presence of p16 or hypo‐phosphorylated pocket proteins (Alevizopoulos et al., 1997). Furthermore, cellular senescence could also be overcome by downregulating Bmi‐1, which suppresses the expression of p16 and p14 at the INK4A locus (Itahana et al., 2003). In contrast, the overexpression of Bmi‐1 results in telomerase activation (Dimri et al., 2002).

5. STRATEGIES OF IMMORTALISATION

Immortalised cells can be defined as a cell population that indefinitely divides and retain their parental characteristics. The amniotic‐derived HAEC and HAMSC can typically be passaged up to passage 5 when expanded in vitro (Murphy et al., 2010; Portmann‐Lanz et al., 2006; Teng et al., 2013). To increase their proliferative capacity, different immortalisation strategies have been developed. These include the overexpression of viral oncogenes (E6/E7), hTERT knock‐in and a combination of pRb knock‐out and hTERT knock‐in (Lipps et al., 2013; Terai et al., 2005; Zhou et al., 2013). These genes interfere with the cell cycle by inhibiting the p53 and Rb pathway (Cascio, 2001; Choi & Lee, 2015) resulting in overcoming cellular senescence and enhancing cellular proliferation.

5.1. Usage of viral oncogenes

Several viral oncogenes are exploited for immortalising primary cells. These include the simian virus 40 large T antigens, human papillomavirus type16 E6 and E7 (HPV‐16 E6/E7), adenoviral E1A/E1B and Tax oncogene in HTLV‐1 (Zheng, 2010). These viral oncogenes interfere with the cell cycle by interfering with the p16/pRb and p53 pathways (Table 1). Notably, only HPV‐16 E6/E7 has been used to successfully establish immortalised cell lines of HAEC and HAMSC (Teng et al., 2013; Zhou et al., 2013). This suggests that HPV‐16 E6/E7 is sufficient to overcome growth arrest of cultured cells, which survive to over 100 passages.

Table 1.

Summary of the functions and immortalisation of in vitro established human cell lines

Cell line Immortalising technique Function Reference
HAEC

‐ Viral oncogenes E6/E7

‐ hTERT

‐ Combination of both
  • ‐ The cells proliferated over 200 PD.
  • ‐ Showed expression of stem cell markers (Oct3/4, Nanog, Sox2, Klf4) and epithelial markers (CD5, CK18)
  • ‐ Showed expression of mesenchymal (CD44, CD73, CD90, CD105) and somatic (CD24, CD29, CD271, Nestin) stem cell markers.
  • ‐ They showed adipogenic, osteogenic, neuronal and cardiac differentiation abilities after induction.
 Zhou et al. (2013)
HAMSC

‐ HPV‐16 E6/E7

‐ Bmi‐1

‐ Combination with hTERT
  • ‐ The cells proliferated over 200 PD.
  • ‐ Showed positive results for CD73, CD90, CD105 and CD44.
  • ‐ Showed negative results for CD34, CD14, CD45 and HLA‐DR.
  • ‐ Successfully expressed stem cell markers like Oct3/4, Sox2, Nanog, Klf4, SSEA4, c‐myc, vimentin and nestin.
  • ‐ They showed adipogenic, osteogenic and chondrogenic differentiation abilities after induction.
 Teng et al. (2013)
Amnion mesenchymal stem cell hTERT
  • ‐ The cells proliferated up to 87 PD.
  • ‐ No changes in surface, marker profile, morphology, karyotype and immunosuppressive capacity.
 Wolbank et al. (2009)
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Abbreviations: Bmi‐1, B lymphoma Mo‐MLV insertion region 1 homolog; CD, cluster of differentiation; HAEC, human amiotic epithelial cells; HAMSC, human amniotic mysenchymal stem cells; hTERT, human telomerase reverse transcriptase; Klf4, Kruppel‐like factors; PD, population doubling; PEPCK, phosphoenolpyruvate carboxykinase; OCT, organic cation transporter; Rb, retinoblastoma; SSEA, stage‐specific embryonic antigen.

5.2. Human telomerase reverse transcriptase

HTERT lengthens the telomeres in DNA strands, thereby avoiding cellular senescence (Shampay & Blackburn, 1988). Primary HAEC and HAMSC were immortalised using knock‐in of the hTERT gene (Teng et al., 2013; Zhou et al., 2013) (Table 1). Immortalised HAECs did not exhibit tumourigenic properties (Zhou et al., 2013). However, one out of two immortalised HAMSC showed increased immunogenicity with altered surface marker profile (Wolbank et al., 2009). Therefore, it is essential to investigate the properties of immortalised cell lines created before being used for further research to avoid unwanted and potential side effects.

5.3. Bmi‐1

A cellular proto‐oncogene c‐myc was found to induce hTERT activity and immortalise human mammary epithelial cells and fibroblasts (Wang et al., 1998). Bmi‐1, a polycomb group of genes, was cloned as an oncogene that controlled the c‐Myc in murine lymphomas (Haupt et al., 1991; Van Lohuizen et al., 1991). However, the introduction of Bmi‐1 altered hTERT activity depending on cells derived from different tissue origins. Introduction of Bmi‐1, weakened the telomerase activity in human epithelial cells but no telomerase activity was found in small airway epithelial cells and dermal keratinocytes (Haga et al., 2007). Alternatively, the suppression of p16 expression was reported when Bmi‐1 was overexpressed in immortalised human mammary epithelial cells, mouse embryonic fibroblasts and HAMSC (Dimri et al., 2002; Haga et al., 2007; Jacobs, Scheijen, et al., 1999; Teng et al., 2013) (Table 1). However, the expression of Bmi‐1 in oral keratinocytes effectively increased its lifespan without significant reduction of p16 expression (Kim et al., 2007). This approach extended the replicative lifespan of human fibroblasts but did not result in immortalisation (Dimri et al., 2002; Jacobs, Kieboom, et al., 1999). Thus suggesting Bmi‐1 overexpression acts differently on hTERT and p16 function depending on cell type.

5.4. Combination approaches

Evidence suggests hTERT alone was insufficient to inhibit the p16/pRb pathway in umbilical cord blood‐derived mesenchymal stem cells and human placenta‐derived mesenchymal cells (Terai et al., 2005). Various combinations of HPV‐16 E6/E7, Bmi‐1 and hTERT have since been used to immortalise amnion‐derived stem cells (Teng et al., 2013; Zhou et al. 2013) (Table 1). Although Bmi‐1 and HPV‐16 E6/E7 alone were sufficient for the inactivation of the p16/pRb pathway, success rates increased when Bmi‐1 and HPV‐16 E6/E7 were combined (Okamoto et al., 2002; Takeda et al., 2004). Thus, indicating effective downregulation of p16 and p53 expression and activation of hTERT is critical to overcoming cellular senescence in different cell types.

6. IMMORTALISED CELL APPLICATION: EXPECTATIONS AND CONCERNS

Immortalised amniotic‐derived HAEC and HAMSC may offer an unlimited supply of pathogen‐free and well‐characterised cells, which could be used in several clinical applications, as well as for research purposes. Currently, no studies have been made on immortalised amniotic‐derived cells for clinical applications. As the immortalised HAEC and HAMSC retain their multi‐lineage differentiation potential (Teng et al., 2013; Zhou et al., 2013) (Figure 5) while maintaining the same anti‐immunogenic and anti‐fibrotic properties as their primary HAEC and HAMSC counterparts, it is plausible that they may be safely applied clinically. Diseases like multiple sclerosis (Mohyeddin Bonab et al., 2007), Crohn's fistula (García‐Olmo et al., 2005), and graft‐versus‐host disease (Ringdén et al., 2006) might be targeted and cured by using immortalised cell lines. However, concern remains about whether these immortalised cells will proliferate uncontrollably once implanted. Some groups have reported that amnion‐derived immortalised HAECs and HAMSCs do not form a tumour when treated into nude mice focusing on testis, muscles and liver (Teng et al., 2013; Zhou et al., 2013). This might provide a promising path that these cells might be used for various regenerative and clinical applications. Yet, more detailed and long‐term validation studies are required on the tumorigenic and immunogenic properties of the immortalised HAECs and HAMSCs before they can be used for further clinical applications (Table 1).

Figure 5.

Figure 5

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Differentiation of immortalised HAMSC and HAEC. (a) The immortalised HAECs have been differentiated into adipocyte, osteocyte, neurons and cardiomyocyte whereas (b) immortalised HAMSC has been differentiated into adipocyte, osteocyte and chondrocyte. HAEC, human amnion epithelial cell; HAMSC, human amniotic mesenchymal stem cell.

7. CONCLUSION

In vitro expansion of HAEC and HAMSC derived from amniotic membranes have gained substantial attention in the regenerative medicine sector. Although, primary HAEC and HAMSC derived from the amniotic membrane have numerous attractive properties as already mentioned; they have limited expansion potential. Accordingly, efforts are being made to make these cells immortal. It is worthwhile to examine other strategies such as blocking p16 and pRB activity to overexpress cell cycle regulators (Nguyen, 2005). The novel immortalisation of the amnion‐derived cells may result in the production of cell lines that could be exploited in regenerative medicine. Moreover, immortalised HAECs and HAMSCs might provide a promising clinical application offering diverse properties that are like primary cell lines. Also, there is a need to resolve some major obstacles like translation from research to good manufacturing practice scale, market authorisation and clinical applications (Table 2).

Table 2.

Advantages and disadvantages of normal and immortalised HAMSC and HAECs

Advantages Disadvantages
Primary HAEC and HAMSC
  • ‐ Biologically accepted
  • ‐ Easily available from discarded placenta
  • ‐ Abundant cells isolated to perform cell therapies
  • ‐ Possess stem cell characteristics therefore maintaining a wider application.
  • ‐ Possesses immunomodulatory properties which can be clinically applicable.
  • ‐ Do not possess tumorigenic properties.

‐ Inability to grow after few passages.

‐ Variable doubling rates and viability

‐ Variable karyotype.

‐ Genetic instability as cells are isolated from different donors.
Immortalised HAEC and HAMSC
  • ‐ Achieving robust cell lines
  • ‐ Short doubling time.
  • ‐ Relatively high consistent viability
  • ‐ Stable karyotype
  • ‐ Maintain original phenotype

‐ Debated biological relevance

‐ Potentially tumorigenic.
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Abbreviations: HAEC, human amnion epithelial cell; HAMSC, human amniotic mesenchymal stem cell.

AUTHOR CONTRIBUTIONS

Aneesa Ansari: conceptualisation, investigation, writing—original draft. Kate M. Denton: writing—review and editing. Rebecca Lim: writing—review and editing.

CONFLICTS OF INTEREST

The authors declare no conflicts of interest.

ACKNOWLEDGEMENTS

1.

This study is supported by Monash Graduate Scholarship and Monash International Postgraduate Research Scholarship—Monash University. Open access publishing facilitated by Monash University, as part of the Wiley‐Monash University agreement via the Council of Australian University Librarians.

Ansari, A. , Denton, K. M. , & Lim, R. (2022). Strategies for immortalisation of amnion‐derived mesenchymal and epithelial cells. Cell Biology International, 46, 1999–2008. 10.1002/cbin.11892

DATA AVAILABILITY STATEMENT

Data sharing not applicable—no new data generated.

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