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Necrotizing enterocolitis (NEC) is a devastating gastrointestinal disease that occurs in neonates. It is one of the most common disease-related causes of death among low birth weight babies, and the mortality rate associated with NEC may be as high as 30% [1,2,3]. Survivors often experience severe complications such as short bowel syndrome and neurological sequelae that negatively impact quality of life [4, 5]. It is reported that hypoxemia, intestinal ischemia, immaturity, and other factors may trigger the inflammatory cascade that correlates with NEC [1]. In the last decade, the intestinal microbiota has been shown to play a role in NEC. Increased levels of Proteobacteria and decreased levels of Firmicutes were identified in NEC patients [6,7,8]. However, the direct link between the microbiota and NEC remains unknown. Causal relationships between the microbiota and host immunity can be strongly informed through the use of germ-free (GF) animal models, and have been widely used to study a variety of diseases such as obesity and depression [9, 10]. In this study, fecal microbial transplantation (FMT) was performed using GF mice to explore the direct link between fecal microbiota and NEC.

HW, XZ, YH and JY conceived and designed the study. LL, FL, QA and YH managed subject recruitment, data and sample collection and sample processing. BZ, HD and XS designed and conducted germ-free mice experiments. WD, DL, CS and JH designed and conducted immune experiments. YH and WD generated and analyzed data. YH, SX and JY wrote the manuscript. All authors read the manuscript and provided critical comments. All authors read and approved the final manuscript.

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Human-induced pluripotent stem cells (hiPSCs) are considered an ideal resource for regenerative medicine because of their ease of access and infinite expansion ability. To satisfy the sizable requirement for clinical applications of hiPSCs, large-scale, expansion-oriented, xeno-free, and cost-effective media are critical. Although several xeno-free media for hiPSCs have been generated over the past decades, few of them are suitable for scalable expansion of cultured hiPSCs because of their modest potential for proliferation and high cost.

Our xeno-free ON2 was compatible with various matrices and ideal for long-term expansion and maintenance of not only healthy-derived hiPSCs but also patient-specific hiPSCs. This highly efficient medium enabled the rapid expansion of hiPSCs in a reliable and cost-effective manner and could act as a promising tool for disease modeling and large-scale production for regenerative medicine in the future.

Therefore, this study was conducted to develop a xeno-free medium that is compatible with various substrates. We developed a versatile ON2/AscleStem hiPSC medium (shortened to ON2) for hiPSCs, which is xeno-free and compatible with multiple matrices. This medium was effective for the stable maintenance of not only healthy hiPSC lines but also patient-derived hiPSC lines. Coupled with lower cost, the developed xeno-free and chemical ON2 medium holds promise for scaled-up production of hiPSCs for drug screening, disease modeling, and regenerative applications.

For long-term expansion, hiPSCs were grafted to prepared substrates with ON2 and seeding densities were adjusted to meet the subculture frequency of once a week on iMatrix-511, FN, and VN or twice on GNF. The most used serum-free medium mTeSR1 and xeno-free media E8 and AK02N were used as positive controls in our study. hiPSCs were maintained at 37 °C with 5% CO2 and 5% O2, and the culture media were changed daily.

To validate the feasibility of ON2 for long-term feeder-free propagation of hiPSCs, we first maintained a 253G1 hiPSC line in ON2 on different matrices, including the iMatrix-511 and GNF. Though both matrices fit for single-cell passage that is beneficial for quality control during hiPSC long-term expansion, enzyme-free dissociation buffer (ethylenediaminetetraacetic acid, EDTA/PBS-based buffer) was especially applied for GNF, which greatly reduces cell damage induced by the enzymatic dissociation buffer and improves quality control (Additional file 2: Fig. S1) [21, 28]. As a positive medium control, we chose xenogeneic mTeSR1 as neither E8 nor AK02N, two common xeno-free media, could support the survival of hiPSCs on GNF, even in the presence of 10 µM Rho-associated protein kinase inhibitor (ROCKi) Y27632, which is known to prevent cell apoptosis induced by dissociation (Additional file 2: Fig. S2a).

Furthermore, 253G1 hiPSCs grown on GNF maintained a normal karyotype (46 XX) without chromosomal abnormalities, similar to those on iMarix-511, which was confirmed by G-banding karyotype analysis (Fig. 2a). This indicated the ability of xeno-free ON2 to maintain the genomic stability of hiPSCs after long-term expansion.

We developed a cost-effective and xeno-free medium, ON2, which was compatible with various substrates including iMatrix-511, FN, VN, and GNF, and which sustained the survival, self-renewal, and pluripotency of hiPSCs, even after prolonged expansion. When compared to other common commercial xeno-free media, ON2 effectively promoted proliferation and maintained the pluripotency of cultured hiPSCs. Moreover, ON2 could sustain not only healthy donor-derived hiPSC lines including 253G1 and ACS-1020 but also the HLA-homo Ff-l01 and the DS patient-specific hiPSC line ACS-1003.

Laminin is reported to be essential for early embryonic development and is widely distributed in various tissues. It has become popular as a natural scaffold for the derivation, expansion, and differentiation of hiPSCs, from laboratory research to clinical trials [9]. Laminin-511 is one of the most commonly used laminin isoforms for sustaining hESCs, and its developed recombinant E8 fragments, commercialized as iMatrix-511, showed high adhesion and support for hiPSCs compared to other ECM proteins combined with various xenogeneic or xeno-free media [18]. Similarly, in our work, we demonstrated that iMatrix-511 showed better cell adhesion and stability with ON2 than other tested commercial matrices [38]. In addition, the single-cell passage and enzyme-free dissociation method and lower ROCKi requirement enhanced the efficiency and stability of cell expansion [39].

Culture medium is another critical component of the culture system, as it provides various growth factors and an optimal required and suitable pH microenvironment for maintaining hiPSCs. In this study, apart from our ON2, we also applied three commercial media including xenogeneic mTeSR1 and xeno-free E8 and AK02N, all of which are commonly used media for hESC/hiPSCs in routine study. mTeSR1 is a complex medium containing several growth factors and signaling agonists that promote hESC growth by stimulating their self-renewal signaling pathway [24, 26]. Although it is xenogeneic, mTeSR1 has been widely applied to diverse culture systems from feeder to feeder-free systems for hESC/hiPSC propagation [15, 25]. In our study, mTeSR1 stably maintained the pluripotency and differentiation potential of hiPSCs on both iMatrix-511 and low-attached GNF (Fig. 1 and Additional file 2: Fig. S2b). Furthermore, mTeSR1 seems to enhance the expression level of NANOG and OCT3/4 of grown hiPSCs, while ON2 improved KLF4 expression specifically.

The simplest xeno-free E8 contains only four growth factors but no albumin, and it is sufficient for the maintenance of hiPSCs combined with different matrices such as VN and iMatrix-511. In our study, 253G1 hiPSCs grown in E8 showed lower proliferation and were prone to spontaneous differentiation over 20 passages (Additional file 2: Fig. S5a). Furthermore, chromosomal abnormality was detected by karyotype analysis (Additional file 2: Fig. S5b), but such morphological variation was not found in the Ff-l01 cells grown in E8 within 15 passages (Additional file 2: Fig. S6). The behavior of E8 may vary between cell lines. However, considering that cells in AK02N and ON2 did not have the above abnormality under the same conditions, we speculate that the lower pluripotency of cells in E8 was related to the lack of albumin, which was in both AK02N and ON2 (Additional file 1: Table S1). It has been reported that albumin can neutralize acidosis, which is responsible for cell differentiation, so we speculated that albumin is essential for the long-term expansion of undifferentiated cells [42]. In this study, the albumin-contained AK02N is efficient and capable for the stable expansion of hiPSCs maintained on iMatrix-511 and no spontaneous differentiation was discovered after propagation (Fig. 3). While comparing to ON2, AK02N displayed less positive to some pluripotent markers such as TRA-1-60, and moreover, it is not sufficient for the cells grown on the low-attached GNF substrate (Additional file 2: Fig. S2a).

Growth factor-based medium is a major focus of hiPSC research, and most popular commercial media, such as mTeSR1 and E8, contain the two factors FGF2 and TGFβ, which drive hPSC pluripotency by activating PI3K/AKT/mTOR pathways and TGFb signaling pathways, respectively, but the high cost of these factors remains a challenge (Additional file 1: Table S1) [24, 26, 27]. We have made some effort toward developing components to control the cost of ON2. On the one hand, we removed TGFβ and reduced the dose of FGF2, which is a thermally unstable growth factor that is usually included in media at a higher dose than is needed. Instead, we supplemented the medium with the cost-effective activin A, which functions similar to TGFβ, LIF that seems to be necessary for more pluripotent state cells, and albumin that acts as a multifaceted antioxidant [42, 46, 52,53,54,55,56]. Although ON2 is more cost-effective than commercial media, it is still dependent on the thermally unstable factor FGF2. Recently, a negligible cost and chemically defined medium for hiPSCs was developed (B8). Compared to E8 and ON2, B8 includes fewer growth factors, including thermostable but more potent FGF2 variants, to reduce the cost [57]. The combination of E8 and Matrigel will not be suitable for clinical applications. AKIT, a growth factor-free medium, is stable for hiPSC culture and is more cost-effective because the expensive and unstable proteins that are included in most culture media have been removed [58]. AKIT medium sustained hiPSCs at the expense of decreased proliferation and poor single-cell survival, claimed by the authors. Although these media are not perfect, they offer novel options to improve ON2 so that it can be more suitable for large-scale expansion of hiPSCs in the future. Besides, further research is needed to meet the quality and safety standards of Good Manufacturing Practices. 2b1af7f3a8