An in vitro model of early anteroposterior organization during human development.

The body plan of the mammalian embryo is shaped through the process of gastrulation, an early developmental event that transforms an isotropic group of cells into an ensemble of tissues that is ordered with reference to three orthogonal axes1. Although model organisms have provided much insight into this process, we know very little about gastrulation in humans, owing to the difficulty of obtaining embryos at such early stages of development and the ethical and technical restrictions that limit the feasibility of observing gastrulation ex vivo2. Here we show that human embryonic stem cells can be used to generate gastruloids-three-dimensional multicellular aggregates that differentiate to form derivatives of the three germ layers organized spatiotemporally, without additional extra-embryonic tissues. Human gastruloids undergo elongation along an anteroposterior axis, and we use spatial transcriptomics to show that they exhibit patterned gene expression. This includes a signature of somitogenesis that suggests that 72-h human gastruloids show some features of Carnegie-stage-9 embryos3. Our study represents an experimentally tractable model system to reveal and examine human-specific regulatory processes that occur during axial organization in early development.

Human gastrulation: The embryo and its models.

Technical and ethical limitations create a challenge to study early human development, especially following the first 3 weeks of development after fertilization, when the fundamental aspects of the body plan are established through the process called gastrulation. As a consequence, our current understanding of human development is mostly based on the anatomical and histological studies on Carnegie Collection of human embryos, which were carried out more than half a century ago. Due to the 14-day rule on human embryo research, there have been no experimental studies beyond the fourteenth day of human development. Mutagenesis studies on animal models, mostly in mouse, are often extrapolated to human embryos to understand the transcriptional regulation of human development. However, due to the existence of significant differences in their morphological and molecular features as well as the time scale of their development, it is obvious that complete knowledge of human development can be achieved only by studying the human embryo. These studies require a cellular framework. Here we summarize the cellular, molecular, and temporal aspects associated with human gastrulation and discuss how they relate to existing human PSCs based models of early development.

Alternative Routes to Induce Naïve Pluripotency in Human Embryonic Stem Cells.

Human embryonic stem cells (hESCs) closely resemble mouse epiblast stem cells exhibiting primed pluripotency unlike mouse ESCs (mESCs), which acquire a naïve pluripotent state. Efforts have been made to trigger naïve pluripotency in hESCs for subsequent unbiased lineage-specific differentiation, a common conundrum faced by primed pluripotent hESCs due to heterogeneity in gene expression existing within and between hESC lines. This required either ectopic expression of naïve genes such as NANOG and KLF2 or inclusion of multiple pluripotency-associated factors. We report here a novel combination of small molecules and growth factors in culture medium (2i/LIF/basic fibroblast growth factor + Ascorbic Acid + Forskolin) facilitating rapid induction of transgene-free naïve pluripotency in hESCs, as well as in mESCs, which has not been shown earlier. The converted naïve hESCs survived long-term single-cell passaging, maintained a normal karyotype, upregulated naïve pluripotency genes, and exhibited dependence on signaling pathways similar to naïve mESCs. Moreover, they undergo global DNA demethylation and show a distinctive long noncoding RNA profile. We propose that in our medium, the FGF signaling pathway via PI3K/AKT/mTORC induced the conversion of primed hESCs toward naïve pluripotency. Collectively, we demonstrate an alternate route to capture naïve pluripotency in hESCs that is fast, reproducible, supports naïve mESC derivation, and allows efficient differentiation.

A systematic analysis of the suitability of preimplantation genetic diagnosis for mitochondrial diseases in a heteroplasmic mitochondrial mouse model.

STUDY QUESTION: What is the reliability of preimplantation genetic diagnosis (PGD) based on polar body (PB), blastomere or trophectoderm (TE) analysis in a heteroplasmic mitochondrial mouse model? SUMMARY ANSWER: The reliability of PGD to determine the level of mitochondrial DNA (mtDNA) heteroplasmy is questionable based on either the first or second PB analysis; however, PGD based on blastomere or TE analysis seems more reliable. WHAT IS KNOWN ALREADY: PGD has been suggested as a technique to determine the level of mtDNA heteroplasmy in oocytes and embryos to avoid the transmission of heritable mtDNA disorders. A strong correlation between first PBs and oocytes and between second PBs and zygotes was reported in mice but is controversial in humans. So far, the levels of mtDNA heteroplasmy in first PBs, second PBs and their corresponding oocytes, zygotes and blastomeres, TE and blastocysts have not been analysed within the same embryo. STUDY DESIGN, SIZE AND DURATION: We explored the suitability of PGD by comparing the level of mtDNA heteroplasmy between first PBs and metaphase II (MII) oocytes (n = 33), between first PBs, second PBs and zygotes (n = 30), and between first PBs, second PBs and their corresponding blastomeres of 2- (n = 10), 4- (n = 10) and 8-cell embryos (n = 11). Levels of mtDNA heteroplasmy in second PBs (n = 20), single blastomeres from 8-cell embryos (n = 20), TE (n = 20) and blastocysts (n = 20) were also compared. PARTICIPANTS/MATERIALS, SETTING, METHODS: Heteroplasmic mice (BALB/cOlaHsd), containing mtDNA mixtures of BALB/cByJ and NZB/OlaHsd, were used in this study. The first PBs were biopsied from in vivo matured MII oocytes. The ooplasm was then subjected to ICSI. After fertilization, second PBs were biopsied and zygotes were cultured to recover individual blastomeres from 2-, 4- and 8-cell embryos. Similarly, second PBs were biopsied from in vivo fertilized zygotes and single blastomeres were biopsied from 8-cell stage embryos. The remaining embryo was cultured until the blastocyst stage to isolate TE cells. Polymerase chain reaction followed by restriction fragment length polymorphism was performed to measure the level of mtDNA heteroplasmy in individual samples. MAIN RESULTS AND THE ROLE OF CHANCE: Modest correlations and wide prediction interval [PI at 95% confidence interval (CI)] were observed in the level of mtDNA heteroplasmy between first PBs and their corresponding MII oocytes (r(2) = 0.56; PI = 45.96%) and zygotes (r(2) = 0.69; PI = 37.07%). The modest correlations and wide PI were observed between second PBs and their corresponding zygotes (r(2) = 0.65; PI = 39.69%), single blastomeres (r(2) = 0.42; PI = 48.04%), TE (r(2) = 0.26; PI = 54.79%) and whole blastocysts (r(2) = 0.40; PI = 57.48%). A strong correlation with a narrow PI was observed among individual blastomeres of 2-, 4- and 8-cell stage embryos (r(2) = 0.92; PI = 11.73%, r(2) = 0.86; PI = 18.85% and r(2) = 0.85; PI = 21.42%, respectively), and also between TE and whole blastocysts (r(2) = 0.90; PI = 23.58%). Moreover, single blastomeres from 8-cell stage embryos showed a close correlation and an intermediate PI with corresponding TE cells (r(2) = 0.81; PI = 28.15%) and blastocysts (r(2) = 0.76; PI = 36.43%). LIMITATIONS, REASONS FOR CAUTION: These results in a heteroplasmic mitochondrial mouse model should be further verified in patients with mtDNA disorders to explore the reliability of PGD. WIDER IMPLICATIONS OF THE FINDINGS: To avoid the transmission of heritable mtDNA disorders, PGD techniques should accurately determine the level of heteroplasmy in biopsied cells faithfully representing the heteroplasmic load in oocytes and preimplantation embryos. Unlike previous PGD studies in mice, our results accord with PGD results for mitochondrial disorders in humans, and question the reliability of PGD using different stages of embryonic development. TRIAL REGISTRATION NUMBER: Not applicable.

Treatment of human embryos with the TGFß inhibitor SB431542 increases epiblast proliferation and permits successful human embryonic stem cell derivation.

STUDY QUESTION: Is there an effect of the TGFß inhibitor SB431542 (SB) on the epiblast compartment of human blastocysts, and does it affect subsequent human embryonic stem cell (hESC) derivation? SUMMARY ANSWER: SB increases the mean number of NANOG-positive cells in the inner cell mass (ICM), and allows for subsequent hESC derivation. WHAT IS KNOWN ALREADY: It is known that inhibition of TGFß by SB has a positive effect on mouse ESC self-renewal, while active TGFß signalling is needed for self-renewal of primed ESC. STUDY DESIGN, SIZE, DURATION: From December 2011 until March 2012, 263 donated spare embryos were used from patients who had undergone IVF/ICSI in our centre. PARTICIPANTS/MATERIALS, SETTING, METHODS: Donated human embryos were cultured in the presence of SB or Activin A, and immunocytochemistry was performed on Day 6 blastocysts for NANOG and GATA6. Moreover, blastocysts were used for the derivation of hESC, with or without exposure to SB. MAIN RESULTS AND THE ROLE OF CHANCE: Immunocytochemistry revealed a significantly higher number of NANOG-positive ICM cells in the SB group compared with the control (12.0 ± 5.9 versus 6.1 ± 4.7), while no difference was observed in the Activin A group compared with other groups (6.7 ± 3.7). The number of GATA6-positive ICM cells did not differ between the SB, Activin A and control group (8.8 ± 4.3, 8.0 ± 4.6 and 7.2 ± 4.0, respectively). Blocking TGFß signalling did not prevent subsequent hESC line derivation. LIMITATIONS, REASONS FOR CAUTION: The number of human blastocysts available for this study was too low to reveal if the observed increase in NANOG-positive epiblast cells after exposure to SB affected the efficiency of hESC derivation (12.5% compared with 16.7%). WIDER IMPLICATIONS OF THE FINDINGS: This work can contribute to the derivation of naive hESC lines in the future. STUDY FUNDING/COMPETING INTEREST(S): M.V.d.J. is holder of a Ph.D. grant of the Agency for Innovation by Science and Technology (IWT, grant number SB093128), Belgium. G.D. and this research are supported by the Research Foundation Flanders (FWO), grant number FWO-3G062910) and a Concerted Research Actions funding from BOF (Bijzonder Onderzoeksfonds University Ghent, grant number BOF GOA 01G01112). S.M.C.d. S.L. is supported by the Netherlands Organization of Scientific Research (NWO) (ASPASIA 015.007.037) and the Interuniversity Attraction Poles (PAI) (no. P7/07). P.D.S. is holder of a fundamental clinical research mandate by the FWO. We would like to thank Ferring Company (Aalst, Belgium) for financial support of this study. The authors do not have any competing interests to declare. TRIAL REGISTRATION NUMBER: Not applicable.

Human surface ectoderm and amniotic ectoderm are sequentially specified according to cellular density.

Mechanisms specifying amniotic ectoderm and surface ectoderm are unresolved in humans due to their close similarities in expression patterns and signal requirements. This lack of knowledge hinders the development of protocols to accurately model human embryogenesis. Here, we developed a human pluripotent stem cell model to investigate the divergence between amniotic and surface ectoderms. In the established culture system, cells differentiated into functional amnioblast-like cells. Single-cell RNA sequencing analyses of amnioblast differentiation revealed an intermediate cell state with enhanced surface ectoderm gene expression. Furthermore, when the differentiation started at the confluent condition, cells retained the expression profile of surface ectoderm. Collectively, we propose that human amniotic ectoderm and surface ectoderm are specified along a common nonneural ectoderm trajectory based on cell density. Our culture system also generated extraembryonic mesoderm-like cells from the primed pluripotent state. Together, this study provides an integrative understanding of the human nonneural ectoderm development and a model for embryonic and extraembryonic human development around gastrulation.

Extensive NEUROG3 occupancy in the human pancreatic endocrine gene regulatory network.

OBJECTIVE: Mice lacking the bHLH transcription factor (TF) Neurog3 do not form pancreatic islet cells, including insulin-secreting beta cells, the absence of which leads to diabetes. In humans, homozygous mutations of NEUROG3 manifest with neonatal or childhood diabetes. Despite this critical role in islet cell development, the precise function of and downstream genetic programs regulated directly by NEUROG3 remain elusive. Therefore, we mapped genome-wide NEUROG3 occupancy in human induced pluripotent stem cell (hiPSC)-derived endocrine progenitors and determined NEUROG3 dependency of associated genes to uncover direct targets. METHODS: We generated a novel hiPSC line (NEUROG3-HA-P2A-Venus) where NEUROG3 is HA-tagged and fused to a self-cleaving fluorescent VENUS reporter. We used the CUT&RUN technique to map NEUROG3 occupancy and epigenetic marks in pancreatic endocrine progenitors (PEP) that were differentiated from this hiPSC line. We integrated NEUROG3 occupancy data with chromatin status and gene expression in PEPs as well as their NEUROG3-dependence. In addition, we investigated whether NEUROG3 binds type 2 diabetes mellitus (T2DM)-associated variants at the PEP stage. RESULTS: CUT&RUN revealed a total of 863 NEUROG3 binding sites assigned to 1263 unique genes. NEUROG3 occupancy was found at promoters as well as at distant cis-regulatory elements that frequently overlapped within PEP active enhancers. De novo motif analyses defined a NEUROG3 consensus binding motif and suggested potential co-regulation of NEUROG3 target genes by FOXA or RFX transcription factors. We found that 22% of the genes downregulated in NEUROG3-/- PEPs, and 10% of genes enriched in NEUROG3-Venus positive endocrine cells were bound by NEUROG3 and thus likely to be directly regulated. NEUROG3 binds to 138 transcription factor genes, some with important roles in islet cell development or function, such as NEUROD1, PAX4, NKX2-2, SOX4, MLXIPL, LMX1B, RFX3, and NEUROG3 itself, and many others with unknown islet function. Unexpectedly, we uncovered that NEUROG3 targets genes critical for insulin secretion in beta cells (e.g., GCK, ABCC8/KCNJ11, CACNA1A, CHGA, SCG2, SLC30A8, and PCSK1). Thus, analysis of NEUROG3 occupancy suggests that the transient expression of NEUROG3 not only promotes islet destiny in uncommitted pancreatic progenitors, but could also initiate endocrine programs essential for beta cell function. Lastly, we identified eight T2DM risk SNPs within NEUROG3-bound regions. CONCLUSION: Mapping NEUROG3 genome occupancy in PEPs uncovered unexpectedly broad, direct control of the endocrine genes, raising novel hypotheses on how this master regulator controls islet and beta cell differentiation.

Rfx6 promotes the differentiation of peptide-secreting enteroendocrine cells while repressing genetic programs controlling serotonin production.

OBJECTIVE: Enteroendocrine cells (EECs) of the gastro-intestinal tract sense gut luminal factors and release peptide hormones or serotonin (5-HT) to coordinate energy uptake and storage. Our goal is to decipher the gene regulatory networks controlling EECs specification from enteroendocrine progenitors. In this context, we studied the role of the transcription factor Rfx6 which had been identified as the cause of Mitchell-Riley syndrome, characterized by neonatal diabetes and congenital malabsorptive diarrhea. We previously reported that Rfx6 was essential for pancreatic beta cell development and function; however, the role of Rfx6 in EECs differentiation remained to be elucidated. METHODS: We examined the molecular, cellular, and metabolic consequences of constitutive and conditional deletion of Rfx6 in the embryonic and adult mouse intestine. We performed single cell and bulk RNA-Seq to characterize EECs diversity and identify Rfx6-regulated genes. RESULTS: Rfx6 is expressed in the gut endoderm; later, it is turned on in, and restricted to, enteroendocrine progenitors and persists in hormone-positive EECs. In the embryonic intestine, the constitutive lack of Rfx6 leads to gastric heterotopia, suggesting a role in the maintenance of intestinal identity. In the absence of intestinal Rfx6, EECs differentiation is severely impaired both in the embryo and adult. However, the number of serotonin-producing enterochromaffin cells and mucosal 5-HT content are increased. Concomitantly, Neurog3-positive enteroendocrine progenitors accumulate. Combined analysis of single-cell and bulk RNA-Seq data revealed that enteroendocrine progenitors differentiate in two main cell trajectories, the enterochromaffin (EC) cells and the Peptidergic Enteroendocrine (PE) cells, the differentiation programs of which are differentially regulated by Rfx6. Rfx6 operates upstream of Arx, Pax6 and Isl1 to trigger the differentiation of peptidergic EECs such as GIP-, GLP-1-, or CCK-secreting cells. On the contrary, Rfx6 represses Lmx1a and Tph1, two genes essential for serotonin biosynthesis. Finally, we identified transcriptional changes uncovering adaptive responses to the prolonged lack of enteroendocrine hormones and leading to malabsorption and lower food efficiency ratio in Rfx6-deficient mouse intestine. CONCLUSION: These studies identify Rfx6 as an essential transcriptional regulator of EECs specification and shed light on the molecular mechanisms of intestinal failures in human RFX6-deficiencies such as Mitchell-Riley syndrome.

Comparative analysis of naive, primed and ground state pluripotency in mouse embryonic stem cells originating from the same genetic background.

Mouse embryonic stem cells (mESCs) exist in a naive, primed and ground state of pluripotency. While comparative analyses of these pluripotency states have been reported, the mESCs utilized originated from various genetic backgrounds and were derived in different laboratories. mESC derivation in conventional LIF + serum culture conditions is strain dependent, with different genetic backgrounds potentially affecting subsequent stem cell characteristics. In the present study, we performed a comprehensive characterization of naive, primed and ground state mESCs originating from the same genetic background within our laboratory, by comparing their transcriptional profiles. We showed unique transcriptional profiles for naive, primed and ground state mESCs. While naive and ground state mESCs have more similar but not identical profiles, primed state mESCs show a very distinct profile. We further demonstrate that the differentiation propensity of mESCs to specific germ layers is highly dependent on their respective state of pluripotency.

Cellular Heterogeneity in the Level of mtDNA Heteroplasmy in Mouse Embryonic Stem Cells.

Variation in the level of mtDNA heteroplasmy in adult tissues is commonly seen in patients with a mixture of wild-type and mutant mtDNA. A mixture of different mtDNA variants may influence such variation and cause mtDNA segregation bias. We analyzed cellular heterogeneity in embryonic stem cells (ESCs) derived from a polymorphic mouse model containing NZB and BALB mtDNA genotypes. In ESCs, inter-colony heterogeneity varied up to 61%, whereas intra-colony heterogeneity varied up to 100%. Three out of five cell lines displayed nearly homoplasmic BALB and NZB mtDNA haplotypes in differentiated single cells. The proportion of NZB mtDNA genotype increased with progressive passaging (0.39%; p = 0.002). These results demonstrate the bimodal segregation of mtDNA haplotypes, indicating the occurrence of tissues with variable levels of heteroplasmies in individuals with mtDNA mutations. Furthermore, proliferation of one mtDNA genotype over another may pose the risk of accumulating mutant mtDNAs during subsequent cell divisions.

Inhibition of transforming growth factor ß signaling promotes epiblast formation in mouse embryos.

Early lineage segregation in preimplantation embryos and maintenance of pluripotency in embryonic stem cells (ESCs) are both regulated by specific signaling pathways. Small molecules have been shown to modulate these signaling pathways. We examined the influence of several small molecules and growth factors on second-lineage segregation of the inner cell mass toward hypoblast and epiblast lineage during mouse embryonic preimplantation development. We found that the second-lineage segregation is influenced by activation or inhibition of the transforming growth factor (TGF)ß pathway. Inhibition of the TGFß pathway from the two-cell, four-cell, and morula stages onward up to the blastocyst stage significantly increased the epiblast cell proliferation. The epiblast formed in the embryos in which TGFß signaling was inhibited was fully functional as demonstrated by the potential of these epiblast cells to give rise to pluripotent ESCs. Conversely, activating the TGFß pathway reduced epiblast formation. Inhibition of the glycogen synthase kinase (GSK)3 pathway and activation of bone morphogenetic protein 4 signaling reduced the formation of both epiblast and hypoblast cells. Activation of the protein kinase A pathway and of the Janus kinase/signal transducer and activator of transcription 3 pathway did not influence the second-lineage segregation in mouse embryos. The simultaneous inhibition of three pathways--TGFß, GSK3ß, and the fibroblast growth factor (FGF)/extracellular signal-regulated kinases (Erk)--significantly enhanced the proliferation of epiblast cells than that caused by inhibition of either TGFß pathway alone or by combined inhibition of the GSK3ß and FGF/Erk pathways only.

Application Of Small Molecules Favoring Naïve Pluripotency during Human Embryonic Stem Cell Derivation.

In mice, inhibition of both the fibroblast growth factor (FGF) mitogen-activated protein kinase kinase/extracellular-signal regulated kinase (MEK/Erk) and the Wnt signaling inhibitor glycogen synthase-3ß (GSK3ß) enables the derivation of mouse embryonic stem cells (mESCs) from nonpermissive strains in the presence of leukemia inhibitory factor (LIF). Whereas mESCs are in an uncommitted naïve state, human embryonic stem cells (hESCs) represent a more advanced state, denoted as primed pluripotency. This burdens hESCs with a series of characteristics, which, in contrast to naïve ESCs, makes them not ideal for key applications such as cell-based clinical therapies and human disease modeling. In this study, different small molecule combinations were applied during human ESC derivation. Hereby, we aimed to sustain the naïve pluripotent state, by interfering with various key signaling pathways. First, we tested several combinations on existing, 2i (PD0325901 and CHIR99021)-derived mESCs. All combinations were shown to be equally adequate to sustain the expression of naïve pluripotency markers. Second, these conditions were tested during hESC derivation. Overall, the best results were observed in the presence of medium supplemented with 2i, LIF, and the noncanonical Wnt signaling agonist Wnt5A, alone and combined with epinephrine. In these conditions, outgrowths repeatedly showed an ESC progenitor-like morphology, starting from day 3. Culturing these "progenitor cells" did not result in stable, naïve hESC lines in the current conditions. Although Wnt5A could not promote naïve hESC derivation, we found that it was sustaining the conversion of established hESCs toward a more naïve state. Future work should aim to distinct the effects of the various culture formulations, including our Wnt5A-supplemented medium, reported to promote stable naïve pluripotency in hESCs.

Assessment of nuclear transfer techniques to prevent the transmission of heritable mitochondrial disorders without compromising embryonic development competence in mice.

To evaluate and compare mitochondrial DNA (mtDNA) carry-over and embryonic development potential between different nuclear transfer techniques we performed germinal vesicle nuclear transfer (GV NT), metaphase-II spindle-chromosome-complex (MII-SCC) transfer and pronuclear transfer (PNT) in mice. No detectable mtDNA carry-over was seen in most of the reconstructed oocytes and embryos. No significant differences were seen in mtDNA carry-over rate between GV NT (n=20), MII-SCC transfer (0.29 ± 0.63; n=21) and PNT (0.29 ± 0.75; n=25). Blastocyst formation was not compromised after either PNT (88%; n=18) or MII-SCC transfer (86%; n=27). Further analysis of blastomeres from cleaving embryos (n=8) demonstrated undetectable mtDNA carry-over in all but one blastomere. We show that NT in the germ line is potent to prevent transmission of heritable mtDNA disorders with the applicability for patients attempting reproduction.

The combination of inhibitors of FGF/MEK/Erk and GSK3ß signaling increases the number of OCT3/4- and NANOG-positive cells in the human inner cell mass, but does not improve stem cell derivation.

In embryonic stem cell culture, small molecules can be used to alter key signaling pathways to promote self-renewal and inhibit differentiation. In mice, small-molecule inhibition of both the FGF/MEK/Erk and the GSK3ß pathways during preimplantation development suppresses hypoblast formation, and this results in more pluripotent cells of the inner cell mass (ICM). In this study, we evaluated the effects of different small-molecule inhibitors of the FGF/MEK/Erk and GSK3ß pathway on embryo preimplantation development, early lineage segregation, and subsequent embryonic stem cell derivation in the humans. We did not observe any effect on blastocyst formation, but small-molecule inhibition did affect the number of OCT3/4- and NANOG-positive cells in the human ICM. We found that combined inhibition of the FGF/MEK/Erk and GSK3ß pathways by PD0325901 and CHIR99021, respectively, resulted in ICMs containing significantly more OCT3/4-positive cells. Inhibition of FGF/MEK/Erk alone as well as in combination with inhibition of GSK3ß significantly increased the number of NANOG-positive cells in blastocysts possessing good-quality ICMs. Secondly, we verified the influence of this increased pluripotency after 2i culture on the efficiency of stem cell derivation. Similar human embryonic stem cell (hESC) derivation rates were observed after 2i compared to control conditions, resulting in 2 control hESC lines and 1 hESC line from an embryo cultured in 2i conditions. In conclusion, we demonstrated that FGF/MEK/Erk and GSK3ß signaling increases the number of OCT3/4- and NANOG-positive cells in the human ICM, but does not improve stem cell derivation.

Effect of light emitting diodes in the photodynamic therapy of rheumatoid arthritis.

BACKGROUND: Complex and painful surgical removal of synovium was replaced by arthroscopic synovectomy as an early treatment of rheumatoid arthritis (RA), which being limited to bigger joints, was replaced by laser synovectomy. Having been more time consuming, laser photodynamic therapy (PDT) replaced this method. Due to thermal side effects of laser PDT, an alternative source of light has been sought. Therefore, to make RA treatment cheaper, less hazardous and suitable according to anatomical geometry, light emitting diodes (LEDs) were used in this study as a potential source of light. METHODS: Red, white, yellow and infra-red (IR) LEDs were tested to measure the optical penetration for soft tissue and their scattering. In vitro study of the cellular response of normal and inflamed lymphocytes from healthy and RA patients was conducted respectively. Methotrexate was injected as photosensitizer to achieve cell-specific precision. RESULTS: IR LEDs showed the maximum penetration and least scattering of all LEDs used. Specimen with drug administration and with subsequent exposure to IR LEDs exhibited massive suppression of inflamed activated lymphocytes in comparison to other controls. CONCLUSION: The properly selected wavelength and intensity of light beam were incident with great precision so that they would not affect unwanted cells, but inflamed activated cells were suppressed due to intense light energy following Methotrexate injection. Without invasion, IR LED PDT showed an effective and cheaper treatment solution for RA.